JP2009517361A - Keratin-binding effector molecule and method for producing the same - Google Patents

Abstract

The present invention relates to a method for producing keratin-binding effector molecules, intermediate products and final products of the method of the invention, and the use of keratin-binding effector molecules produced by said method in skin cosmetics. The invention further relates to a method of using a skin cosmetic active ingredient on the skin and / or hair and a method of increasing the time for which the active substance stays on the skin and hair.
[Selection figure] None

Description

  The present invention relates to a method for producing keratin-binding effector molecules, as well as intermediates and final products of the method of the invention, and the use of keratin-binding effector molecules produced according to the invention in skin cosmetics. Furthermore, the invention relates to a method for using a skin cosmetic active ingredient on the skin and / or hair and a method for increasing the residence time of the active ingredient on the skin and hair.

  Vertebrate cells contain filaments, a group of which is composed of keratin. For example, certain proteins such as desmoplakin or plakophilin bind to these keratins in the hair, skin and fingernails and toenails by means of specific sequence motifs, so-called keratin binding domains (Fontao L, Favre B, Riou S, Geerts D, Jaunin F, Saurat JH, Green KJ, Sonnenberg A, Borradori L., Interaction of the bullous pemphigoid antigen 1 (BP230) and desmoplakin with intermediate filaments is mediated by distinct sequences within their COOH terminus., Mol Biol Cell. 2003 May; 14 (5): 1978-92.Epub 2003 Jan 26; Hopkinson SB, Jones JC., The N-terminus of the transmembrane protein BP180 interacts with the N-terminal domain of BP230, thus mediating keratin cytoskeleton anchorage to the cell surface at the site of the hemidesmosome, Mol Biol Cell.2000 Jan; 11 (1): 277-86; Smith EA, Fuchs E., Defining the Interactions Between Intermediate Filaments and Desmosomes, The Journal of Cell Biology , Vo lume 141, 1998).

  Human skin is exposed to some kind of aging effect, partly due to its intrinsic action (aging with time) and partly due to external factors (aging due to the environment, for example light). In addition, skin conditions such as acne, greasy or dry skin, photosensitivity reactions such as keratosis, rosacea, dermatoses and photodermatoses, inflammatory reactions, erythema reactions, allergic reactions or autoimmune reactions Temporary or persistent changes in appearance can occur.

  External factors include, inter alia, artificial light sources of irradiation having sunlight or a similar spectrum, and free radicals or ionic compounds that may be generated as a result of irradiation. These factors include tobacco smoke, and reactive compounds found in tobacco smoke, such as ozone, free radicals, singlet oxygen and other active oxygen or nitrogen compounds that disrupt the natural physiology or tissue of the skin. Also mentioned.

  In Germany, total ozone has declined by just under 10% since 1968, or about 3% in 10 years. During the same period, UV increased by about 15%. UV-B radiation (wavelength about 300 nm), which causes sunburn, has the most cancer effects. This increases the risk of suffering from so-called non-melanoma skin cancer (squamous cell carcinoma or squamous cell carcinoma or basal cell carcinoma or basal cell carcinoma). In this regard, tumor risk increases with the number of sunburns. In particular, UV exposure (sunburn in children) during the first decade of life affects the risk of cancer.

  According to WHO estimates, 2 million people worldwide have basal cell carcinoma and squamous cell carcinoma of the skin each year, and approximately 200,000 people have melanoma. In Germany, there are approximately 120,000 new cases of skin cancer, 7% of which are melanoma. In Germany, approximately 1600 people die each year from melanoma or non-melanoma skin cancer (Arztezeitung 5.17.2000).

  The need for new active ingredients and products, and innovative methods of applying them, to prevent and treat the above disorders, diseases and also to care and cosmetic treatment of skin, hair, fingernails and toenails Sex is steadily increasing.

  A German patent application with application reference number DE102005011988.3 describes the use of keratin binding domains in cosmetic preparations. In the international patent application having the application reference number PCT / EP / 05/005599 it is also disclosed that the keratin binding domain can be coupled with an effector molecule.

  One object of the present invention was to provide a new type of skin cosmetic active ingredient compound for application to the skin, hair, fingernails and toenails, as well as a method of producing the same. Advantageously, it has been found that the active ingredient compounds have keratin binding properties and are suitable for producing cosmetic and / or skin cosmetic formulations or preparations. Furthermore, it was an object of the present invention to identify suitable compounds that can be covalently coupled to polypeptides having keratin binding properties. In particular, it was an object of the present invention to provide an innovative method of using skin cosmetic active ingredients. Furthermore, it was an object to provide a method for increasing the residence time of the skin cosmetic active ingredient on the skin, hair and / or fingernails and toenails.

In a first embodiment, the present invention provides an effector molecule (i) bearing at least one hydroxy or amino function on a keratin binding polypeptide (ii), a thioester bond, an ester bond, a thioether bond. A method of generating a keratin-binding effector molecule by coupling using a linker molecule (iii) having at least two coupling functional groups capable of a bond selected from the group consisting of an ether bond and an amide bond,
(a) in the first coupling step, the effector molecule (i) is first bound to the linker molecule (iii) via an ester bond or an amide bond;
(b) In a further coupling step, the reaction product from (a) is coupled to the keratin binding polypeptide (ii) via the still free coupling functionality of the linker molecule (iii).

  In a further embodiment of the invention, the coupling according to the invention between the linker molecule (iii) and the effector molecule (i) takes place via an esterification reaction mediated by carbodiimide or acid chloride.

  In a preferred embodiment of the invention, the effector molecule (i) used in the method of the invention is selected from the group consisting of pigments, photoprotective agents, vitamins, provitamins, carotenoids, antioxidants and peroxide degrading agents. The

  In a particularly preferred embodiment, a keratin-binding polypeptide (ii) is used that has binding affinity for human skin, hair or nail keratin.

Preferably, the keratin-binding polypeptide (ii) used according to the invention is
(a) SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 146, 150, 153 , 156, 157, 158, 160, 162, 164, 166, 168 or 170, or
(b) SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 146, 150, 153 , 156, 157, 158, 160, 162, 164, 166, 168 or 170, comprising a polypeptide that is at least 40% identical and capable of binding to keratin.

Preferably, the keratin-binding polypeptide (ii) used according to the invention has a binding affinity for keratin of human skin, hair or nails,
(a) SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 146, 150, 153 156, 157, 158, 160, 162, 164, 166, 168 or 170, a nucleic acid molecule encoding a polypeptide comprising the sequence shown in
(b) SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 145, 149, 152 159, 161, 163, 165, 167 or 169, a nucleic acid molecule comprising at least one polynucleotide of the sequence shown in
(c) SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96 , 98 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 146, 150, 153, A nucleic acid molecule encoding a polypeptide according to 156, 157, 158, 160, 162, 164, 166, 168 or 170,
(d) SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 145, 149, 152 , 159, 161, 163, 165, 167 or 169, or a nucleic acid molecule corresponding to at least one of the sequences according to SEQ ID NO: 2, 4, derived from this nucleic acid molecule by substitution, deletion or insertion 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, Nucleic acid molecule encoding a polypeptide that is at least 40% identical to at least one of the sequences according to 146, 150, 153, 156, 157, 158, 160, 162, 164, 166, 168 or 170 and capable of binding to keratin ,
(e) a nucleic acid molecule encoding a polypeptide recognized by a monoclonal antibody against the polypeptide encoded by the nucleic acid molecule according to (a)-(c),
(f) a nucleic acid molecule encoding a keratin-binding protein that hybridizes with the nucleic acid molecule according to (a) to (c) under stringent conditions,
(g) Under stringent hybridization conditions, at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt, the nucleic acid molecule according to (a) to (c) or a partial fragment thereof is used as a probe. A nucleic acid molecule encoding a keratin-binding protein, which can be isolated from a DNA bank
(h) SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 146, 150, 153 156, 157, 158, 160, 162, 164, 166, 168 or 170, a nucleic acid molecule that can be generated by back-translating one of the amino acid sequences shown in
Preferably it can be encoded by a nucleic acid molecule comprising at least one nucleic acid molecule selected from the group consisting of:

  In one embodiment of the invention, it is preferred to use a linker molecule (iii) having at least two different coupling functional groups. Preferably these are linker molecules (iii) carrying maleimide groups.

In the method of the present invention, the linker molecule (iii) particularly preferably used is represented by the general formula 1:

(Wherein “n” is an integer from 0 to 20).

  In the most preferred embodiment of the method of the invention, maleimidocaproic acid is used as the linker molecule (iii).

In a further preferred embodiment of the invention this is:
(i) the keratin-binding polypeptide used is SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, Including one of the sequences shown in 138, 140, 146, 150, 153, 156, 157, 158, 160, 162, 164, 166, 168 or 170,
(j) the linker molecule (iii) used is maleimidocaproic acid, and
(k) The effector molecule (i) is selected from the group consisting of pantothenic acid, panthenol, an ester of panthenol, an ether of panthenol, and a cation-derivatized panthenol.

  The present invention also provides that the effector molecule (i) is indirectly coupled to the keratin-binding polypeptide via the linker molecule (iii), provided that the linker molecule (iii) is not maleimide but keratin-binding polypeptide ( ii) does not correspond to SEQ ID NO: 166 and the effector molecule (ii) relates to a keratin-binding effector molecule that is not a fluorescent dye. In a preferred embodiment, it is a keratin-binding polypeptide (ii), SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, A polypeptide or protein comprising one of the sequences according to 134, 136, 138, 140, 146, 150, 153, 156, 157, 158, 160, 162, 164, 166, 168 or 170, a linker molecule (iii) Maleimide caproic acid is used as a keratin bond further comprising an effector molecule (i) selected from the group consisting of pantothenic acid, panthenol, esters of panthenol, ethers of panthenol, and cation-derivatized panthenol Effe It is a ter molecule.

  The present invention further provides the use of the above-described keratin-binding effector molecule of the present invention in skin cosmetics, and the particularly preferred skin cosmetics are skin protection compositions, skin care compositions, skin cleansing compositions, hair protection compositions. Hair care compositions, hair cleaning compositions, hair colorants, fingernails and toenails care compositions, and decorative cosmetics.

Furthermore, the present invention provides a method for applying a skin cosmetic active ingredient to skin, hair, and / or nail keratin, wherein:
(l) coupling the skin cosmetic active ingredient to a keratin-binding polypeptide; and
(m) The keratin-binding effector molecule according to (k) is applied to the skin, hair and / or nail keratin as a component of a skin cosmetic preparation.

Furthermore, the present invention provides a method for increasing the residence time of a skin cosmetic active ingredient in skin, hair and / or nail keratin, wherein:
(n) coupling a skin cosmetic active ingredient to a keratin-binding polypeptide;
(o) applying the keratin-binding effector molecule according to (m) to skin, hair and / or nail keratin as a component of a skin cosmetic preparation; and
(p) The active ingredient binds indirectly to the skin, hair or fingernails or toenails via the keratin binding domain.

The present invention relates to Formula 2:

Further provided is a compound wherein “n” corresponds to an integer from 0 to 20.

  The present invention further provides a skin cosmetic comprising a keratin-binding effector molecule produced by the above method, wherein the keratin-binding polypeptide (ii) does not correspond to SEQ ID NO: 166.

Definitions “Antibodies” for the purposes of the present invention are proteins produced by humans and vertebrates with jaws to protect against antigens (infectious pathogens or biological materials that are foreign to the body). Antibodies are central components in the higher eukaryotic immune system and are secreted by B cells, a type of white blood cell. Antibodies are in the blood and in extracellular fluids of tissues.

  “Reverse translation” for the purposes of the present invention means the translation of a protein sequence into a nucleic acid sequence encoding the protein. Accordingly, reverse translation is a method of decoding an amino acid sequence into a corresponding nucleic acid sequence. Traditional methods are based on the creation of codon usage tables for specific organisms obtained by computer-based sequence comparisons. A codon usage table can be used to determine the most frequently used codons for a particular amino acid of a particular organism. Protein reverse translation is known to those skilled in the art and can be performed using computer algorithms specifically created for this purpose (Andres Moreira and Alejandro Maass.TIP: protein backtranslation aided by genetic algorithms. Bioinformatics, Volume 20, Number 13 Pp. 2148-2149 (2004); G Pesole, M Attimonelli, and S Liuni.A backtranslation method based on codon usage strategy.Nucleic Acids Res. 1988 March 11; 16 (5 Pt A): 1715-1728).

  “Decorative cosmetics” means cosmetic aids that are not used primarily for care, but are used to beautify or improve the appearance of skin, hair, and / or fingernails and toenails To do. This type of auxiliaries is well known to those skilled in the art, for example, coal pencil, mascara, eye shadow, colored day cream, powder, concealer stick, blusher, lipstick, lip liner stick, makeup, nail polish Liquid, glamor gel, and the like. Compositions suitable for coloring the skin or hair are also included.

  “Skin cosmetics”, also referred to as “medicinal cosmetics” or “skin cosmetic compositions” or “skin cosmetic preparations”, refers to (i) skin, hair, and / or fingernails and toenails. (Ii) to treat existing damage to skin, hair, and / or fingernails or toenails, and (iii) skin, hair, and / or fingernails Or compositions or preparations for the care of toenails, skin cosmetics, nail cosmetics, hair cosmetics, skin, hygiene or pharmaceutical compositions, preparations and formulations, and skin (sensory properties) ) Is included. Decorative cosmetic compositions are expressly included. A skin care composition is also included, and a dermatological purpose of use as a drug is realized in view of a cosmetic aspect. This type of composition or preparation is used to help, prevent and treat skin disorders, producing biological effects in addition to cosmetic effects. For the purposes of the above definition, “skin cosmetics” are well known to those skilled in the art in cosmetically compatible media and are described in cosmetic handbooks such as Schrader, Grundlagen und Rezepturen der Kosmetika [Fundamentals and formulations. of cosmetics], Huthig Verlag, Heidelberg, 1989, ISBN 3-7785-1491-1, or Umbach, Kosmetik: Entwicklung, Herstellung und Anwendung kosmetischer Mittel [Cosmetics: development, manufacture and use of cosmetic compositions], 2nd extended edition, 1995 , Georg Thieme Verlag, ISBN 3 13 712602 9, including suitable auxiliaries and additives.

  “Skin cosmetic active ingredient” or “skin cosmetic active ingredient” for the purposes of the present invention means in the skin cosmetic according to the above definition, which is concerned with realizing the individual mechanism of action of the skin cosmetic. It is an active ingredient. Thus, for example, they protect skin, hair, and / or fingernails or toenails damage, and (ii) treat existing damage to skin, hair, and / or fingernails and toenails (Iii) has the property of caring for skin, hair, and / or fingernails or toenails, and (iv) decorative beautification or skin, hair, and / or fingers Active ingredient used to improve the appearance of nails and toenails. Active ingredients for skin care are also included, and the dermatological purpose of use as a drug is realized in consideration of a cosmetic viewpoint. This type of active ingredient is used to help, prevent and treat skin disorders and provides biological effects in addition to cosmetic effects. Active ingredients of this type are for example natural or synthetic polymers, pigments, wetting agents, oils, waxes, proteins, enzymes, minerals, vitamins, sunscreens, dyes, fragrances, antioxidants, peroxide degrading agents, preservatives Used to help, prevent, and treat skin damage, and have biological effects that restore health, prevent damage, regenerate, or improve the overall condition of the skin Selected from the group of pharmaceutically active ingredients.

An “effector molecule” for the purposes of the present invention is a specific foreseeable effect on skin, hair, and / or fingernails or toenails, preferably biological or physiological protective effects, preventive effects. And / or a skin or skin cosmetic active ingredient that has a care and / or cosmetic effect. The effector molecules are preferably dyes, photoprotective agents, vitamins, proteins, enzymes, provitamins, antioxidants, peroxide decomposers and compounds containing fatty acids, conditioners or metal ions, very particularly preferably group A, Non-protein derived compounds such as vitamins from group B, C and E, vitamins, provitamins and vitamin precursors, with vitamins B ₁ , B ₂ and B ₅ being particularly preferred. Very particular preference is given to pantothenic acid, panthenol and panthenol derivatives, in particular panthenol esters and ethers, and cation-derivatized panthenol is also very particularly preferred.

  “Increased residence time of skin cosmetic active ingredient on skin, hair, and / or fingernails or toenails” refers to residence time compared to active ingredient not coupled to keratin-binding polypeptide. Is prolonged in time, thus increasing the effectiveness of this active ingredient in the skin and / or hair. Preferably, the increase in residence time on the skin, hair, and / or fingernails or toenails is compared to the other uncoupled active ingredients under the same use conditions. 10%, 15%, 20%, particularly preferably 30%, 40%, 50%, very particularly preferably 75% of the active ingredient present on the hair and / or fingernails or toenails %, 100%, 125%, most preferably 150%, 200%, 300%, most preferably all of which means an increase of 500%, 750%, 1000%.

  “Keratin” for the purposes of the present invention means an intermediate filament composed of a rope-like protein complex. The intermediate filament is composed of many proteins of the same type (monomer) arranged in parallel and has a tube-like structure. The intermediate filaments combine to form a relatively large bundle (tension fiber). Intermediate filaments, together with microtubules and actin filaments, form the cytoskeleton of the cell. There are five types of intermediate filaments. Acid keratin and basic keratin, desmin, neurofilament and lamin. Particularly preferred for the purposes of the present invention are acidic keratins and basic keratins in the epithelium (monolayer or multilayer cell layer covering all the outer body surface of a multicellular animal organism). One or more “keratins” (or stratum corneum, hard proteins) refer to proteins involved in cell stability and shape. This protein is a component of mammalian skin, hair and nails. The strength of keratin is enhanced by fiber formation. Each amino acid chain forms a right-handed α-helix, and these helices form a left-handed superhelix (= protofibril) every third. Eleven protofibrils gather to form microfibrils, and these gathers also bundle to form macrofibrils that surround, for example, hair cells.

  “Keratin binding polypeptide” means a polypeptide or protein having the property of binding to keratin within the meaning of the above definition. Thus, keratin-binding polypeptides are also proteins associated with intermediate filaments. These keratin-binding polypeptides have binding affinity for keratin or for macrostructures composed of keratin such as protofibrils, microfibrils or macrofibrils. Furthermore, a keratin-binding polypeptide is understood to mean a polypeptide having binding affinity to mammalian skin, hair, and / or fingernails or toenails.

  A “keratin binding polypeptide” is also a polypeptide having a biological function associated with binding of keratin, keratin fibers, skin or hair in a mammalian organism. Keratin-binding polypeptide also means a binding motif or protein domain that is required for actual binding to keratin, keratin fibers, skin or hair. The binding of keratin-binding polypeptide (ii) to keratin can be tested under the conditions described in Examples 8, 9 and 10. The keratin-binding polypeptide is about 10%, 20% of the keratin-binding ability of desmoplakin (SEQ ID NO: 2), preferably keratin-binding domain B of desmoplakin (SEQ ID NO: 4) in the quantitative keratin binding test described above. 30%, 40% or 50%, preferably 50%, 60%, 70%, 80% or 90%, particularly preferably 100%, 125%, 150%, very particularly preferably 200%, 300% or A polypeptide having 400%, most preferably 500%, 600%, 700% or 1000% or more.

  Cosmetic compositions for oral care, dental care, gum care and denture care for purposes of the present invention are Umbach: Kosmetik, which is expressly incorporated herein by reference, for example, by reference: Entwicklung, Herstellung und Anwendung kosmetischer Mittel [Cosmetics: development, manufacture and use of cosmetic compositions], chapter 7, page 187-219, 2nd expanded edition, 1995, Georg Thieme Verlag, ISBN 3 13 712602 9 Means all compositions, preparations and delivery forms suitable for oral hygiene, dental hygiene, gum hygiene and denture hygiene. These compositions, preparations and delivery forms are well known to those skilled in the art and this list is not considered exhaustive, but includes, for example, toothpastes, toothpastes, toothpastes, children's toothpaste creams, Includes toothpaste gels, liquid toothpaste creams, mouthwashes, mouth washes, ointments and pastes. The production of such compositions is well known to those skilled in the art and is generally known as textbooks (e.g. Umbach: Kosmetik: Entwicklung, Herstellung und Anwendung kosmetischer Mittel [Cosmetics: development, manufacture and use of cosmetic compositions], 2nd expanded edition. , 1995, Georg Thieme Verlag, ISBN 3 13 712602 9). Thus, in addition to keratin-binding effector molecules according to the invention and / or produced by the methods of the invention, these compositions also contain additional components known to those skilled in the art. These may be, for example, surfactants, detergents, active ingredients, binders, wetting agents, viscosity modifiers, preservatives, dyes, fragrances and sweeteners, but this list is not considered exhaustive. The specific active ingredient is preferably an active ingredient used for gum inflammation or oral injury. In addition, these active ingredients may be effective, for example, against plaque bacteria or to protect the gums. Textbooks Umbach: Kosmetik: Entwicklung, Herstellung und Anwendung kosmetischer Mittel [Cosmetics: development, manufacture and use of cosmetic compositions], 2nd expanded edition, 1995, Georg Thieme Verlag, ISBN 3 13 712602 9, pp. 205-207 Are explicitly referred to herein.

  “Cosmetic compatible medium” is to be understood broadly and means substances suitable for the production of cosmetic or dermocosmetic preparations and mixtures thereof. They are preferably media compatible with proteins.

  When in contact with human and / or animal skin tissue or hair, a “cosmetically compatible substance” does not cause irritation or damage and is not incompatible with other substances. In addition, these substances have a slight allergenic potential and have been approved for use in cosmetic preparations by nationally registered accreditation bodies. These substances are well known to those skilled in the art, for example cosmetic handbooks such as Schrader, Grundlagen und Rezepturen der Kosmetika [Fundamentals and formulations of cosmetics], Huthig Verlag, Heidelberg, 1989, ISBN 3-7785-1491-1 Can be found in

  “Nucleic acid” or “nucleic acid molecule” means deoxyribonucleotides, ribonucleotides, or polymers or hybrids thereof in sense or antisense orientation, in single or double stranded form. The term nucleic acid or nucleic acid molecule can be used to describe a gene, DNA, cDNA, mRNA, oligonucleotide or polynucleotide.

  “Nucleic acid sequence” means a continuous, bound sequence of deoxyribonucleotides or ribonucleotides of a nucleic acid molecule as defined above, which can be confirmed using available DNA / RNA sequencing methods, Represented or indicated by a list of abbreviations, letters or words to represent.

  “Polypeptide” for the purposes of the present invention means a macromolecule composed of amino acid molecules in which amino acids are linearly linked via peptide bonds. Polypeptides can be composed of a number of amino acids (about 10-100), but also include proteins that are generally composed of at least 100 amino acids. However, it can contain thousands of amino acids. Preferably, the polypeptide is at least 20, 30, 40 or 50, particularly preferably at least 60, 70, 80 or 90, very particularly preferably at least 100, 125, 150. 175 or 200, most preferably at least 200 amino acids, although an upper limit of several thousand amino acids is possible.

`` Homology '' or `` identity '' between two nucleic acid sequences is understood to mean the identity of the nucleic acid sequence over the entire sequence length of interest, which is the program algorithm GAP (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG), Madison, USA; Altschul et al. (1997) Nucleic Acids Res. 25: 3389ff), calculated by comparison with the following parameter settings:
Gap weight: 50
Length weight: 3
Average match: 10
Average mismatch: 0.

  For example, a sequence having at least 80% homology with SEQ ID NO: 1 based on nucleic acid is a sequence having at least 80% homology when compared to SEQ ID NO: 1 by the above program algorithm with the above set of parameters Is understood to mean.

Homology between two polypeptides is understood to mean amino acid sequence identity over the entire sequence length of interest, which is the program algorithm GAP (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG ), With the assistance of Madison, USA), calculated by comparison with the following parameter settings:
Gap weight: 8
Length weight: 2
Average match: 2.912
Average mismatch: -2.003.

  For example, a polypeptide-based sequence having at least 80% homology with SEQ ID NO: 2 has at least 80% homology when compared to SEQ ID NO: 2 by the above program algorithm with the above set of parameters It is understood to mean an array.

  “Hybridization conditions” should be understood broadly and refer to hybridization conditions that are stringent or less stringent depending on the application. Such hybridization conditions include, among others, Sambrook J, Fritsch EF, Maniatis T et al., In Molecular Cloning (A Laboratory Manual), 2nd edition, Cold Spring Harbor Laboratory Press, 1989, pages 9.31-9.57) or Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6. One skilled in the art will select hybridization conditions that can distinguish between specific and non-specific hybridization. For example, the conditions during the washing step may be selected from low stringency conditions (about 2 × SSC, 50 ° C.) and high stringency conditions (about 0.2 × SSC, 50 ° C., preferably 65 ° C.). (20 × SSC: 0.3M sodium citrate, 3M NaCl, pH 7.0). Furthermore, the temperature during the washing step can be increased from a low stringency condition of about 22 ° C., which is room temperature, to a higher stringency condition of about 65 ° C. Both the salt concentration and temperature parameters can be changed individually, simultaneously or in any case while keeping the other parameters constant. During hybridization, for example, denaturing agents such as formamide or SDS can also be used. Hybridization is preferably performed at 42 ° C. in the presence of 50% formamide. Some exemplary conditions for the hybridization and washing steps are shown below.

1. Hybridization conditions can be selected, for example, from the following conditions:
(a) 4 x SSC, 65 ° C,
(b) 6 x SSC, 45 ° C,
(c) 6 × SSC, 100 μg / ml denatured and fragmented fish sperm DNA, 68 ° C.,
(d) 6 × SSC, 0.5% SDS, 100 μg / ml denatured salmon sperm DNA, 68 ° C.
(e) 6 × SSC, 0.5% SDS, 100 μg / ml denatured and fragmented salmon sperm DNA, 50% formamide, 42 ° C.
(f) 50% formamide, 4 × SSC, 42 ° C., or
(g) 50% (volume / volume) formamide, 0.1% bovine serum albumin, 0.1% ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer (pH 6.5), 750 mM NaCl, 75 mM sodium citrate, 42 ° C., or
(i) 2x or 4x SSC, 50 ° C (low stringent conditions),
(j) 30-40% formamide, 2 × or 4 × SSC, 42 ° C. (low stringent conditions)
500 mN sodium phosphate buffer (pH 7.2, 7% SDS (g / V), 1 mM EDTA, 10 μg / ml single-stranded DNA, 0.5% BSA (g / V) (Church and Gilbert, Genomic sequencing. Proc. Natl. Acad. Sci. USA 81: 1991. 1984).

2. The washing step can be selected, for example, from the following conditions:
(a) 0.015M NaCl / 0.0015M Sodium citrate / 0.1% SDS, 50 ° C
(b) 0.1 x SSC, 65 ° C
(c) 0,1 x SSC, 0.5% SDS, 68 ° C
(d) 0.1 × SSC, 0.5% SDS, 50% formamide, 42 ° C
(e) 0.2 x SSC, 0.1% SDS, 42 ° C
(f) 2 × SSC, 65 ° C. (low stringent conditions).

  In one embodiment, stringent hybridization conditions are selected as follows.

A hybridization buffer containing formamide, NaCl and PEG6000 is selected. The presence of formamide in the hybridization buffer destabilizes the double-stranded nucleic acid molecule, and as a result, the hybridization temperature can be lowered to 42 ° C. without reducing the stringency. The use of a salt in the hybridization buffer increases duplex regeneration or hybridization efficiency. PEG increases the viscosity of the solution, which has an adverse effect on the regeneration rate, and the presence of the polymer in the solution increases the concentration of the probe in the remaining medium, thereby increasing the hybridization rate. . The composition of the buffer is as follows.

  Hybridize overnight at 42 ° C. Wash the filter 3 times the next morning, in each case with 2 x SSC + 0.1% SDS for about 10 minutes.

  In connection with the description, “hydroxy functional group bearing effector molecules”, “hydroxy functional groups” allow molecules bearing these OH groups to be covalently bound to other molecules via esterification reactions. It means a free OH group or a hydroxyl group. “Hydroxy functional groups” for the purposes of the present invention are also those that can be chemically converted to OH functional groups such as derivatives such as methoxy, ethoxy and the like. Here, the effector molecule of the present invention has at least one hydroxyl group. However, it is also possible to use effector molecules having two, three or more hydroxy functional groups.

  In connection with the description, an “amino functional group-carrying effector molecule”, “amino functional group” refers to an amino group that allows the amino functional group-carrying molecule to be covalently bonded to another molecule via an amide bond. means. An “amino functional group” for the purposes of the present invention is also one that can be chemically converted to an amino functional group. Here, the effector molecule of the present invention has at least one amino functional group. However, it is also possible to use effector molecules having two, three or more amino functions and / or secondary amino groups.

  “Coupling” in connection with the binding of a linker molecule to an effector molecule or keratin binding protein means a covalent attachment of said molecule.

  A “coupling functional group” is a functional group of a linker molecule that can be covalently linked to an effector molecule or a functional group of a keratin binding protein. Non-limiting examples that may be mentioned are hydroxy groups, carboxyl groups, thio groups and amino groups. “(Coupling functional group)” or “coupling functional group” and “(multiple) anchor group” or “anchor group” are used synonymously.

The present invention provides an effector molecule (i) bearing at least one hydroxy or amino function on a keratin binding polypeptide (ii) from a thioester bond, an ester bond, a thioether bond, an ether bond and an amide bond. Providing a method of generating a keratin-binding effector molecule by coupling using a linker molecule (iii) having at least two coupling functional groups capable of binding selected from the group consisting of:
(a) in the first coupling step, the effector molecule (i) is first bound to the linker molecule (iii) via an ester bond or an amide bond;
(b) In a further coupling step, the reaction product from (a) is coupled to the keratin binding polypeptide (ii) via the still free coupling functionality of the linker molecule (iii).

  In a particularly preferred embodiment, the linker molecule (iii) has at least two coupling functional groups or anchor groups, at least one of these groups being a carboxyl functional group. The coupling of the linker molecule (iii) to the effector molecule takes place via the carboxyl functional group, and the effector linker molecule is coupled to the keratin binding polypeptide (ii) with the remaining anchor group.

  A preferred linkage of the linker molecule (iii) to the keratin binding polypeptide (ii) is, for example, active via an amino, thiol or hydroxy function, which can form a corresponding amide, thioester or ester bond. After conversion, it is carried out with the carboxyl functional group of the linker molecule (iii) if appropriate.

  In a particularly preferred embodiment of the invention, the linker molecule (iii) has at least two different coupling functional groups, and very particularly preferred herein is a linker molecule (iii) having a maleimide group .

In the process of the invention, the linker molecule (iii) used is particularly preferably of the general formula 1:

Wherein `` n '' is 0-40 or 0-20, preferably 0-15, particularly preferably 0-10, very particularly preferably 1-9, or 2-8, or 3-7, all Among them, a carboxylic acid group-carrying maleimide is most preferably an integer of 5. The use of maleimidocaproic acid is most preferred among all. Furthermore, the use of maleimidocaproic acid chloride is very particularly preferred.

In a further particularly preferred embodiment, the linker molecule (iii) has at least two different coupling functional groups, as well as modules that increase hydrophilicity or lipophilicity. This preferred linker molecule is shown in Formula 1b.

In the formula, “n” is an integer of 0 to 40 or 0 to 20, preferably 0 to 15, particularly preferably 0 to 10, particularly very preferably 1 to 9, or 2 to 8, or 3 to 7. X is a group O, S, N, CH ₂ , —OC═O, O═CO—, —NR, —NR—C═O, O═C—NR—, R is H; methyl , ethyl, propyl, isopropyl, butyl, isobutyl, sec- butyl, tert- butyl, C ₁ pentyl, isopentyl, neopentyl, tert- pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl or the like cycloalkyl, alkyl group not branched or branched -C _12; benzoyl, benzyl; phenyl and naphthyl, C ₆ -C _10, such heteroaryl - aryl group, preferably H, methyl and ethyl, "module", Ethylene glycol or 2-40, preferably 2-20, especially preferred Is a polyethylene glycol group having 2 to 10 repeating units; or an amino acid preferably selected from the group consisting of glycine, alanine, serine, threonine, glutamic acid, glutamine, aspartic acid, asparagine, arginine and cysteine; or 2-40 Polypeptide, preferably 2 to 20, particularly preferably 2 to 10 amino acids (amino acids are preferably polar amino acids, particularly preferably glycine, alanine, serine, threonine, glutamic acid, glutamine, aspartic acid Or selected from the group consisting of asparagine, arginine and cysteine); or a polyacryl having 2 to 100, preferably 2 to 80, particularly preferably 2 to 50, most preferably 2 to 20 monomer units To improve acidity or lipophilicity , “Module” means an alkyl group having 2 to 40 carbon atoms; or a polyolefin group having 2 to 40, preferably 2 to 20, particularly preferably 2 to 10 repeating units, or preferably glycine. , Valine, leucine, isoleucine, phenylalanine, tryptophan, proline, methionine, or a polypeptide having 2 to 40, preferably 2 to 20, particularly preferably 2 to 10 amino acids ( The amino acid is preferably a nonpolar amino acid, particularly preferably selected from the group consisting of glycine, valine, leucine, isoleucine, phenylalanine, tryptophan, proline, methionine), or 2 to 100, preferably 2 to 80 Units, particularly preferably 2 to 50, most preferably 2 to 20 monomer units. Esters, polyamide or polyurethane.

In a further preferred embodiment, the linker molecule has the general formula 1c:

Wherein X at the o, m or p position is COOH or R-COOH, and R is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl , tert- pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, C ₁ -C ₁₂ linear or branched alkyl groups such as dodecyl, or one or more C ₁ -C ₄ - alkyl groups optionally substituted C ₅ -C ₁₂ - cycloalkyl group such as a cycloalkyl group; or o-, m- or p- orientation aryl, benzyl or benzoyl units, by preferably cyclohexyl, phenyl and naphthyl) Is the molecule shown.

  In a further preferred embodiment, R may be a “module” as described in formula 1b.

  In a further preferred embodiment, the coupling of the linker molecule (iii) with the effector molecule (i) described in (a) is a carbodiimide, anhydride or acid chloride mediated esterification reaction or amide formation, The use of an acid chloride of the linker molecule (iii) is particularly preferred. A reaction mediated by a carbodiimide, an anhydride or an acid chloride is a reaction with a carbodiimide or a symmetrical anhydride or mixture required for ester or amide formation between the linker molecule (iii) and the effector molecule (i). It means activation of the carboxyl group of the linker molecule (iii) by a reaction to obtain an anhydride or a reaction to obtain an acid chloride.

  The carbodiimide to be mentioned is preferably dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), N '-(3-dimethylaminopropyl) -N-ethylcarbodiimide hydrochloride (EDC), and the use of diisopropylcarbodiimide or EDC is preferred. Particularly preferred. Furthermore, activation with carbonyldiimidazole (CDI) can be performed. These esterifications are carried out in the presence of 0.1 to 100 mol%, preferably 0.5 to 10%, particularly preferably 1 to 6% of N, N-dimethylaminopyridine (DMAP). Amide formation can be carried out by reacting a compound activated by carbodiimide with an amine. Optionally, amide formation can be performed in the presence of additives such as N-hydroxysuccinimide, pentafluorophenol or N-hydroxybenzotriazole. Such additives are known to those skilled in the art. If these additives give active esters which can be isolated, the reaction of these isolated active esters with the effector molecule is also understood as esterification or amide formation mediated by the carbodiimide according to the invention. Is done.

  The reaction of the linker molecule (iii) to obtain the anhydride is carried out by a general method known to those skilled in the art. For example, the use of mixed anhydrides such as those obtained by reaction with acetic anhydride, pivaloyl anhydride, acetyl chloride, pivaloyl chloride or chloroformate is preferred. Anhydrous pivaloyl and carbonic anhydride are particularly preferred. When using acid chlorides, it is advantageous to carry out the formation of anhydrides in the presence of a tertiary base such as, for example, pyridine, triethylamine.

  Coupling of the linker molecule (iii) with the effector molecule (i) described in (a) may be preferably performed after the activation of the linker molecule (iii), and the anhydride is present in the presence of a base. Is obtained. Preferred bases to mention are aromatic and tertiary alkyl amines such as pyridine, triethylamine, tributylamine, trioctylamine, ethyldiisopropylamine and the like. In a particularly preferred embodiment, the base used is triethylamine.

For the reaction of the linker molecule (iii) with the acid chloride, the chlorinating agent used is a conventional chlorinating agent known to the person skilled in the art, for example thionyl chloride, phosphorus trichloride, phosphorus pentachloride, chloride. Oxalyl, phosgene, or phosphorus oxychloride. Very particular preference is given to using thionyl chloride (SOCl ₂ ).

  Using thionyl chloride, maleimidocaproic acid can be converted to the acid chloride. Suitable solvents here are aromatic and aliphatic hydrocarbons (e.g. benzene, toluene, xylene, hexane, heptane etc.), halogenated hydrocarbons (e.g. methylene chloride), ethers (e.g. diethyl ether, THF etc.) , And excess chlorinating agent itself. In a preferred embodiment, toluene is used. Chlorination can be carried out with or without a catalyst. DMF is particularly preferred as a catalyst for chlorination.

  In a further preferred embodiment, the coupling of the linker molecule (iii) described in (a) with the effector molecule (i) is carried out directly after the activation of the linker molecule (iii) described above, in the presence of a base. The acid chloride is obtained. Preferred bases are aromatic and tertiary alkyl amines such as pyridine, triethylamine, tributylamine, trioctylamine, ethyldiisopropylamine and the like. In a particularly preferred embodiment, the base used is triethylamine.

  Surprisingly, during the acylation of panthenol with maleimidocaproic acid chloride in the presence of triethylamine, selectivity other than carbodiimide-mediated coupling can be achieved with respect to the formation of potential isomers. It was found. During the acylation of panthenol with maleimidocaproic acid chloride in the presence of triethylamine, only two of the three possible monoacylated isomers are formed in a ratio of about 4: 3 (implementation). (See Example 12).

  Thus, the present invention more preferably provides the use of triethylamine as a base catalyst in combination with a linker molecule (iii) that reacts to give an acid chloride, wherein the linker molecule (iii) is particularly preferably maleimidocaproic acid. And the effector molecule (i) is particularly preferably panthenol.

  Optionally, the reaction product from step (a) (hereinafter referred to as linker effector molecule (iv)) can be further purified to separate possible isomers of the reaction product. Here, all conventional methods of purifying chemicals can be used, for example distillation, rectification, crystallization, extraction and chromatographic purification methods. It is preferable to perform column chromatography.

  The reaction product obtained from step (a) above is bound to the keratin-binding polypeptide (ii) via the second, still free anchor group of the linker molecule. For example, such an anchor group can be a thiol functional group, which allows the linker to disulfide bond with the cysteine group of keratin-binding polypeptide (ii).

  The use of tailored linkers allows precise matching of the linker effector molecule (iv) binding to the keratin binding polypeptide. Furthermore, as a result, it is possible to bind two or more effector molecules to the keratin binding polypeptide (ii).

  The linker used depends on the functionality to be coupled. Suitable are for example polypeptide (ii) to keratin bonds by sulfhydryl reactive groups (e.g. maleimide, pyridyl disulfide, α-haloacetyl, vinylsulfone, sulfatoalkylsulfone (preferably sulfatoethylsulfone)). The molecule to be coupled.

  A covalent bond between the linker molecule (iii) and the keratin-binding polypeptide (ii) is preferred. This can be done, for example, via the side chain of the keratin binding polypeptide (ii), in particular via an amino function, a hydroxy function, a carboxylic acid function or a thiol function. One or more amino functions of lysine groups; one or more thiol groups of cysteine groups; one or more hydroxyl groups of serine, threonine or tyrosine groups; one or more carboxyls of aspartic acid or glutamic acid groups Linking via the group; or via the N-terminal or C-terminal functional group of the keratin-binding polypeptide (ii) is preferred. Apart from the amino acid functional group in the primary sequence of the keratin-binding polypeptide (ii), an amino acid having an appropriate functional group (e.g. cysteine, lysine, aspartic acid, glutamic acid) is added to the sequence, or the amino acid of the polypeptide sequence Can also be substituted by such amino acid functional groups. Methods of mutagenesis or nucleic acid molecule manipulation are well known to those skilled in the art. Some selected methods are described below.

  It is particularly preferred to use a linker effector molecule (iv) prepared using maleimidocaproic acid, which is preferred for the method of the present invention. In the case of such a linker effector molecule (iv), the cysteine group present in the keratin binding polypeptide is used for coupling.

  The success of the effector coupling can be monitored by two different tests.

(i) Elman test that can determine the number of free Cys-SH groups in the protein before and after effector coupling. A substantial decrease in free SH groups after coupling indicates that a good reaction has proceeded (see Example 24).

(ii) Activity test that can measure the binding of keratin-binding polypeptide to hair with and without a coupled linker effector molecule (see Example 24a).

  In a further embodiment according to the invention, the binding of the effector molecule is in some sense by the action of an endogenous enzyme (e.g. esterase, lipase or glucosidase) or by ambient conditions on the skin over time (e.g. moisture, acidic pH). In “sustained release” or “controlled release” in such a way that it can be removed and released from the keratin-binding polypeptide (II). Thus, the keratin-binding polypeptide (II) can be used as an application system and can be applied once or repeatedly to provide free effector molecules on the skin in small amounts. Basically, it is known to those skilled in the art that an effector can be released on the skin from its corresponding derivative, for example from tocopherol acetate, ascorbyl palmitate or ascorbyl glucoside (representative literature). : Redoules, D. et al. J. Invest. Dermatol. 125, 2005, 270, Beijersbegen van Henegouwen, GMJ et al., J. Photochem. Photobiol. 29, 1995, 45.).

  In a further preferred embodiment of the present invention, for the method of the present invention, a hydroxyl group selected from the group consisting of pigments, photoprotective agents, vitamins, provitamins, carotenoids, antioxidants and peroxide decomposers, or An effector molecule (i) carrying an amino group is used. Here, the effector molecule used can have one or more hydroxyl or amino groups.

Among pigment pigments, food pigments, semi-permanent pigments, reactive dyes or oxidation dyes are preferred. In the case of oxidative dyes, one component as an effector molecule (i) and a keratin-binding polypeptide sequence (ii) are combined and then oxidized at the site of action, i.e. after binding to the hair, with a second pigment component. Preferably. In the case of oxidative dyes, it is also preferred to couple the pigment component prior to coupling with the keratin binding polypeptide sequence (ii).

  Suitable dyes are basically all conventional hair dyes provided that they have hydroxyl or amino groups that can be coupled. Suitable dyes are known to those skilled in the art from cosmetic handbooks such as Schrader, Grundlagen and Rezepturen der Kosmetika [Fundaments and formulations of cosmetics], Huthig Verlag, Heidelberg, 1989, ISBN 3-7785-1491-1.

  Preferred food dyes are anthocyan, anthocyanidins (pelargonidin, cyanidin, delphinidin, peonidin, petunidin, malvidin), betalains such as betacyan, betaxanthine, carmine, carminic acid, kermesic acid, cochineal red A, hydroxycoumarin (umbelliferone , Esculetin, scopoletin, flaxetine), 2-hydroxy-1,4-naphthoquinone.

Particularly advantageous dyes are those specified in the list below. The color index number (CIN) is adopted from Rowe Color Index, 3rd edition, Society of Dyers and Colorists, Bradford, England, 1971.

  The dyes described above can also be used as effector molecules (i) to the skin or nail binding polypeptide sequence (ii) to stain the skin or nails (eg, tattoos). Particularly suitable are fluorescent dyes (e.g. the fluorescence contained in Table 2) to achieve a healthy and shiny skin tone and to optically lighten the skin color (`` whitening '') after application to the skin. The use of keratin-binding effector molecules containing dyes). The use of fluorescent pigments is described, for example, in US 6,753,002. Fluorescent dyes for obtaining a healthier skin tone are described in "Filling the Fluorescent Palette, Cosmetics & Toiletries, 26-34, 121, No. 5, 2006". For example, fluorescent dyes from DayGlo are preferred. In addition, these keratin-binding effector molecules, including fluorescent dyes, can also be used to lighten the hair color and to give the hair a special reflection or shine. This is described, for example, in “Hair lightening by fluorescent dyes, Cosmetics & Toiletries, 56-57, 120, No. 7, 2005” and in the specification US 2004 / 0,258,641 cited therein.

  Further preferred effector molecules (i) are carotenoids. According to the invention, carotenoid is understood to mean the following compounds and their esterified or glycosylated derivatives: Individually or as a mixture, xanthophylls such as violaxanthin, lutein and zeaxanthin, astaxanthin, capsanthin, capsorubin, cryptoxanthin, bixin, 3-hydroxyechinenone, adonilbin and the like. Carotenoids preferably used are lutein, astaxanthin, zeaxanthin, mutatoxanthin, luteoxanthin and auroxanthin.

  Further preferred effector molecules (i) are vitamins, in particular vitamin A and its esters.

  For the purposes of the present invention, retinoid means vitamin A alcohol (retinol). In this regard, the term retinoic acid includes both all-trans retinoic acid as well as 13-cis-retinoic acid. The term retinol preferably includes all-trans compounds. A preferred retinoid for use in the suspensions of the present invention is all-trans retinol, hereinafter referred to as retinol.

  Further preferred effector molecules (i) are vitamins from groups A, C and E, provitamins and vitamin precursors, especially 3,4-didehydroretinol, ascorbic acid (vitamin C), and palmitate ester, ascorbine Acid glucosides or phosphates, tocopherols, in particular α-tocopherol.

Vitamin E or tocopherol for the purposes of the present invention includes eight fat-soluble derivatives, which are further classified as tocopherols and tocotrienols. The isoprenoid side chain of tocopherol is derived from phytyl pyrophosphate (PP), while tocotrienol has a side chain derived from geranylgeranyl-PP. These subclasses of α, β, γ and δ derivatives differ in the degree of methylation of the 6-chromanol cyclic structure. Tocopherol has a saturated side chain (1) and tocotrienol (2) has an unsaturated side chain.

  In the present invention, vitamin E or tocopherol means all of the above-mentioned tocopherol or tocotrienol. Furthermore, 6-chromanol derivatives can also be used as effector molecules according to the invention.

  Preferred vitamin B group vitamins, provitamins or vitamin precursors or derivatives thereof, and derivatives of 2-furanone that are preferably used according to the invention include, among others:

Vitamin B ₁ , common name thiamine, chemical name 3-[(4′-amino-2′-methyl-5′-pyrimidinyl) methyl] -5- (2-hydroxyethyl) -4-methylthiazolium chloride.

Vitamin B ₂ , common name riboflavin, chemical name 7,8-dimethyl-10- (1-D-ribityl) benzo [g] pteridine-2,4 (3H, 10H) -dione. Riboflavin exists, for example, in free form in whey, and other riboflavin derivatives can be isolated from bacteria and yeast. A riboflavin stereoisomer which is also suitable in the present invention is roxoflavin, which can be isolated from fish meal or liver and has a D-arabityl group instead of D-ribitil.

Vitamin B ₅ (pantothenic acid and panthenol). It is preferred to use panthenol. Panthenol derivatives that can be used according to the invention are in particular esters and ethers of panthenol and cation-derivatized panthenol. In a further preferred embodiment of the invention, in addition to pantothenic acid or panthenol, derivatives of 2-furanone can also be used. Particularly preferred derivatives are also the commercially available substances dihydro-3-hydroxy-4,4-dimethyl-2 (3H) -furanone (Merck), 4-hydroxymethyl-γ, the common name pantolactone. -Butyrolactone (Merck), 3,3-dimethyl-2-hydroxy-γ-butyrolactone (Aldrich) and 2,5-dihydro-5-methoxy-2-furanone (Merck). Clearly includes all stereoisomers.

  Advantageously, these compounds provide moisturizing and skin soothing properties to the keratin-binding effector molecules of the present invention.

Vitamin B ₆ . This is not a uniform substance, but means a derivative of 5-hydroxymethyl-2-methylpyridin-3-ol known by the common names pyridoxine, pyridoxamine and pyridoxal.

  In the present invention, suitable derivatives (salts, esters, sugars, nucleotides, nucleosides, peptides and lipids) of the compounds can be used as effector molecules. Preferred lipophilic, oil-soluble antioxidants from this group are tocopherol and its derivatives, gallic esters, flavonoids and carotenoids, and butylhydroxytoluene / anisole. The flavonoids shown in Table 8 are particularly preferred.

  More preferred are so-called peroxide decomposing agents, ie compounds that can decompose peroxides, particularly preferably lipid peroxides. These are understood to include organic substances such as, for example, 5-pyrimidinol and 3-pyridinol derivatives and probucol.

  Further preferred effector molecules are silicones such as hexamethyldisiloxane, octamethyltrisiloxane, decamethyltetrasiloxane, 1,1,3,3-tetraisopropyldisiloxane, octaphenyltrisiloxane, 1,3,5-trivinyl. -1,1,3,5,5-pentamethyltrisiloxane and the like. In a preferred embodiment, chlorosiloxane is reacted with a compound of formula 1, 1b or 1c to give the corresponding siloxyl ester. Examples of chlorosiloxanes that can be used include chloropentaphenyldisiloxane, 1,3-dichlorotetraphenyldisiloxane, 1,3-dichlorotetramethyldisiloxane, 1,5-dichlorohexamethyltrisiloxane, and the like.

  In a further preferred embodiment, the halomethylsiloxane is reacted with a compound of formula 1, 1b or 1c to give the corresponding methylsiloxyl ester, such as chloromethylpentadisiloxane, chloromethylheptamethylcyclotetrasiloxane, 3-chloromethyl Heptamethyltrisiloxane, 1,3-bis (bromomethyl) tetramethyldisiloxane, 3,5-bis (chloromethyl) octamethyltetrasiloxane, and the like are obtained.

  In a further preferred embodiment, silicones having hydroxy or amino groups are used and can react with compounds of formula 1, 1b or 1c to form esters or amides. Examples of such silicones are 3-aminopropylpentamethyldisiloxane, 3-hydroxypropylpentamethyldisiloxane, 1,1,3,3-tetraphenyldisiloxane diol, 1,3-bis (hydroxybutyl) tetra Such as methyldisiloxane.

  A further preferred effector molecule (i) is a UV light protection filter. These are understood to mean organic substances that absorb ultraviolet light and can release the absorbed energy again in the form of longer wavelength radiation, for example heat. The organic material may be oil-soluble or water-soluble.

Oil-soluble UV-B filters that can be used are, for example, the following substances:
4-aminobenzoic acid derivatives, preferably 2-ethylhexyl 4- (dimethylamino) benzoate having a free NH function, 2-octyl 4- (dimethylamino) benzoate and amyl 4- (dimethylamino) benzoate Derivative.
Esters of salicylic acid, preferably 2-ethylhexyl salicylate, 4-isopropylbenzyl salicylate, homomenthyl salicylate.
Derivatives of benzophenone, preferably 2-hydroxy-4-methoxybenzophenone, 2-hydroxy-4-methoxy-4′-methylbenzophenone, 2,2′-dihydroxy-4-methoxybenzophenone.
An ester of benzalmalonic acid, preferably a derivative of di-2-ethylhexyl 4-methoxybenzmalonate having a free OH function.
Propane-1,3-dione, such as 1- (4-tert-butylphenyl) -3- (4′-methoxyphenyl) propane-1,3-dione.

Suitable water-soluble substances are
A sulfonic acid derivative of benzophenone, preferably 2-hydroxy-4-methoxybenzophenone-5-sulfonic acid and a salt thereof.

  Esters of cinnamic acid, preferably 4-methoxycinnamic acid 2-ethylhexyl, 4-methoxycinnamic acid isopentyl, 2-cyano-3-phenylcinnamic acid 2-ethylhexyl (octocrylene) derivatives having a free OH functional group Is particularly preferred.

  In addition, derivatives of benzophenone, in particular the use of 2-hydroxy-4-methoxybenzophenone, 2-hydroxy-4-methoxy-4'-methylbenzophenone, 2,2'-dihydroxy-4-methoxybenzophenone, and propane-1,3 The use of -diones such as 1- (4-tert-butylphenyl) -3- (4'-methoxyphenyl) propane-1,3-dione is preferred.

A suitable and typical UV-A filter is
Derivatives of benzoylmethane, such as 1- (4'-tert-butylphenyl) -3- (4'-hydroxy-phenyl) propane-1,3-dione, 4-tert-butyl-4'-hydroxydibenzoylmethane Or 1-phenyl-3- (4′-isopropylphenyl) propane-1,3-dione, etc.
An aminohydroxy substituted derivative of benzophenone, such as N, N-diethylaminohydroxybenzoyl-n-hexylbenzoic acid.

  Naturally, the UV-A and UV-B filters can be mixed and used.

Suitable UV filter materials are shown in the table below.

  In addition to the two main photoprotective substances listed above, it is also possible to use antioxidant-type secondary photoprotective agents that interrupt the chain of photochemical reactions that are triggered when UV light penetrates into the skin. . Typical examples are tocopherol (vitamin E) and ascorbic acid (vitamin C).

  A further group is anti-irritants that have anti-inflammatory effects on skin damaged by ultraviolet radiation. Such substances are, for example, bisabolol, phytol and phytantriol.

  A further group of preferred effector molecules are polyphenols. For the purposes of the present invention, polyphenol is a collective name for phenolic compounds belonging to different classes of substances, most of which have more than two phenol groups or phenol ether groups.

(a) Hydroxycinnamic acid, hydroxycoumarin, hydroxybenzoic acid
(b) Catechin, leucoanthocyanidins
(c) Anthocyanidins
(d) Flavonone
(e) Flavon and flavonol.

In the method of the present invention,
(a) SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 146, 150, 153 156, 157, 158, 160, 162, 164, 166, 168 or 170, or
(b) SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 146, 150, 153 156, 157, 158, 160, 162, 164, 166, 168 or 170 and at least 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%, in particular Preferably at least 75%, 80%, 85%, 90%, 91%, 92%, 93% or 94%, very particularly preferably at least 95% or 96% identical and capable of binding to keratin A keratin-binding polypeptide (ii) corresponding to a peptide is preferred.

In a preferred embodiment of the invention, the keratin-binding polypeptide (ii) used is
(a) SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 146, 150, 153 156, 157, 158, 160, 162, 164, 166, 168 or 170, a nucleic acid molecule encoding a polypeptide comprising the sequence shown in
(b) SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 145, 149, 152 , 159, 161, 163, 165, 167, 169, a nucleic acid molecule comprising at least one polynucleotide of the sequence shown in particular, preferably 165 and 167, most preferably 167,
(c) SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 146, 150, 153 156, 157, 158, 160, 162, 164, 166, 168 or 170, a nucleic acid molecule encoding a polypeptide,
(d) SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 145, 149, 152 , 159, 161, 163, 165, 167 or 169, or a nucleic acid molecule corresponding to at least one of the sequences according to SEQ ID NO: 2, 4, derived from this nucleic acid molecule by substitution, deletion or insertion 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, At least one of the sequences according to 146, 150, 153, 156, 157, 158, 160, 162, 164, 166, 168 or 170 and at least 40%, 45% or 50%, preferably at least 55%, 60%, 65% Or 70%, particularly preferably at least 75%, 80%, 85%, 90%, 91%, 92%, 93% or 94%, very particularly preferably at least 95% or 96% identical and bind to keratin A nucleic acid molecule encoding a polypeptide capable of
(e) a nucleic acid molecule encoding a polypeptide recognized by a monoclonal antibody against the polypeptide encoded by the nucleic acid molecule according to (a)-(c),
(f) a nucleic acid molecule encoding a keratin-binding protein that hybridizes with the nucleic acid molecule according to (a) to (c) under stringent conditions,
(g) Using at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt, or a partial fragment thereof according to (a) to (c) as a probe under stringent hybridization conditions A nucleic acid molecule encoding a keratin binding protein, which can be isolated from a DNA bank
(h) SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 146, 150, 153 , 156, 157, 158, 160, 162, 166, 168 or 170, at least one nucleic acid molecule selected from the group consisting of nucleic acid molecules that can be generated by back-translating one of the amino acid sequences shown in It is encoded by the nucleic acid molecule it contains.

  Suitable keratin-binding polypeptide domains according to the present invention are present in the polypeptide sequence of desmoplakin, placofilin, placoglobin, plectin, periplakin, emboplakin, trichohyalin, epiplatin or hair follicles, particularly preferably desmoplakin and placofilin .

  In a preferred embodiment of the invention, desmoplakin according to SEQ ID NO: 2, 42, 44, 46, 48, 146, 150, 153, 156, 157, 158, 160, 162, 164 or 166, or a partial sequence thereof, and / or a sequence Placofilin or a partial sequence thereof according to number 18, 20, 26, 28, 32, 34, 36, 168, 170, and / or SEQ ID NO: 50, 52, 54, 56, 58, 60, 62, 64, 66, 68 , Plakoglobin according to the sequence 70, or a partial sequence thereof, and / or periplakin according to the sequence SEQ ID NO: 86, and / or emboplatin or a partial sequence thereof according to the sequences SEQ ID NO: 90, 92, 94, 96, 98, 102, 104, 105 And / or the sequences according to SEQ ID NO: 138 and 140 are used as keratin-binding polypeptides. Preferred keratin binding domains are desmoplakin polypeptides set forth in SEQ ID NOs: 4, 6, 8, 10, 12, 14, 146, 150, 153, 156, 157, 158, 160, 162, 164, 166, 168 or 170. , And its functional equivalent. In particularly preferred embodiments of the invention, the keratin binding polypeptides shown in SEQ ID NOs: 156, 157, 158, 160, 162, 164, 166, 168 and / or 170 are used in the methods of the invention. In the most preferred embodiment of the invention, the keratin binding protein shown in SEQ ID NO: 168 is used. It goes without saying here that this protein can be used with or without the histidine anchor in SEQ ID NO: 168. Thus, a histidine anchor (or a purification / detection system used similarly) may also be at the C-terminus. In practical use of the keratin-binding protein (eg in cosmetic preparations), histidine anchors (or purification / detection systems used similarly) are not necessary. Therefore, it is preferred to use the protein without an additional amino acid sequence.

The present invention provides a medium that is cosmetically compatible,
(i) SEQ ID NOs: 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110 , 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 146, 150, 153, 156, 157, 158, 160, 162, 164, 166 , A nucleic acid molecule encoding a polypeptide comprising the sequence shown in 168 or 170,
(j) (b) SEQ ID NOs: 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 145, 149, 152, 159, 161, 163, 165, 167 or 169, particularly preferably a nucleic acid molecule comprising at least one polynucleotide of the sequence shown in SEQ ID NO: 165 and 167, most preferably SEQ ID NO: 167,
(k) SEQ ID NOs: 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110 , 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 146, 150, 153, 156, 157, 158, 160, 162, 164, 166 , Nucleic acid molecules encoding polypeptides according to 168 or 170,
(l) SEQ ID NOs: 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109 , 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 145, 149, 152, 159, 161, 163, 165, 167 or 169 A nucleic acid molecule having a nucleic acid sequence corresponding to at least one of the following: or derived from this nucleic acid molecule by substitution, deletion or insertion, SEQ ID NOs: 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 146, 150, 153, 156, 157, 158, 160, 162, 164, 166, 1 At least one of the sequences according to 68 or 170 and at least 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%, particularly preferably at least 75%, 80%, 85%, 90%, A nucleic acid molecule encoding a polypeptide that is 91%, 92%, 93% or 94%, very particularly preferably at least 95% or 96% identical and capable of binding to keratin,
(m) a nucleic acid molecule encoding a polypeptide recognized by a monoclonal antibody against the polypeptide encoded by the nucleic acid molecule according to (a)-(c),
(n) a nucleic acid molecule encoding a keratin binding protein that hybridizes with the nucleic acid molecule according to (a) to (c) under stringent conditions,
(o) Under stringent hybridization conditions, using at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt, the nucleic acid molecule according to (a) to (c) or a partial fragment thereof as a probe A nucleic acid molecule encoding a keratin binding protein, which can be isolated from a DNA bank,
(p) SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 146, 150, 153 , 156, 157, 158, 160, 162, 166, 168 or 170, at least one nucleic acid molecule selected from the group consisting of nucleic acid molecules that can be generated by back-translating one of the amino acid sequences shown Further provided is a cosmetic composition for treating a keratin-containing material comprising at least one keratin-binding polypeptide (ii) encoded by a nucleic acid molecule comprising:

  Suitable keratin-binding polypeptide domains according to the present invention are present in the polypeptide sequence of desmoplakin, placofilin, placoglobin, plectin, periplakin, emboplakin, trichohyalin, epiplakin or hair follicle, particularly preferably desmoplakin and placofilin .

  In a preferred embodiment of the invention, desmoplakin according to SEQ ID NO: 2, 42, 44, 46, 48, 146, 150, 153, 156, 157, 158, 160, 162, 164 or 166, or a partial sequence thereof, and / or a sequence Placofilin or a partial sequence thereof according to number 18, 20, 26, 28, 32, 34, 36, 168, 170, and / or SEQ ID NO: 50, 52, 54, 56, 58, 60, 62, 64, 66, 68 70, plakoglobin or a partial sequence thereof according to the sequence, and / or periplakin according to the sequence of SEQ ID NO: 86, and / or And / or the sequences according to SEQ ID NO: 138 and 140 are used as keratin-binding polypeptides. Preferred keratin binding domains are desmoplakin polypeptides set forth in SEQ ID NOs: 4, 6, 8, 10, 12, 14, 146, 150, 153, 156, 157, 158, 160, 162, 164, 166, 168 or 170 , And its functional equivalent. In particularly preferred embodiments of the invention, the keratin binding polypeptides shown in SEQ ID NOs: 156, 157, 158, 160, 162, 164, 166, 168 and / or 170 are used in the methods of the invention. In the most preferred embodiment of the invention, the keratin binding protein shown in SEQ ID NO: 168 is used. It goes without saying that this protein can be used with or without the histidine anchor in SEQ ID NO: 168. Thus, a histidine anchor (or a purification / detection system used similarly) may also be at the C-terminus. In practical use of the keratin-binding protein (eg in cosmetic preparations), histidine anchors (or purification / detection systems used similarly) are not necessary. Therefore, the use of said protein without further amino acid sequences is preferred.

  The present invention further provides a pharmaceutical composition for treating a keratin-containing material comprising at least one keratin-binding polypeptide (ii) as defined above in a pharmaceutically compatible medium.

  The formulation base of the pharmaceutical composition of the present invention preferably contains a pharmaceutically acceptable adjuvant. Auxiliaries known for use in the pharmaceutical field, food technology and related technologies, especially those listed in the relevant pharmacopoeia (eg DAB Ph. Eur. BP NF) and their properties hinder physiological use No other auxiliaries are pharmaceutically acceptable.

  Also included in the present invention are the “functional equivalents” of the specifically disclosed keratin binding polypeptides (ii) and their use in the methods of the present invention.

  For the purposes of the present invention, a “functional equivalent” or analog of a specifically disclosed keratin-binding polypeptide (ii) is a keratin-binding polypeptide that also has a desired biological activity, such as, for example, keratin binding. Are different polypeptides. Thus, for example, a “functional equivalent” of a keratin-binding polypeptide is the SEQ ID NO: 2, 4, 6, 8, 10 in the quantitative keratin binding test described in the Examples under otherwise equivalent conditions. , 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110 , 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136 138, 140, 146, 150, 153, 156, 157, 158, 160, 162, 164, 166, 168 or 170, preferably SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 40, 42, 44, 46, 48, 146, 150, 153, 156, 157, 158, 160, 162, 164, 166, 168 or 170, particularly preferably 166 and 168, most preferably about 10%, 20%, 30%, 40% or 50% of the keratin binding capacity of the polypeptide shown in 168, Preferably 60%, 70%, 80% or 90%, particularly preferably 100%, 125%, 150%, very particularly preferably 200%, 300% or 400%, most preferably 500%, 600%, It is understood to mean a polypeptide having 700% or 1000% or more.

  According to the present invention, a “functional equivalent” has an amino acid other than those specifically described in at least one sequence position of the amino acid sequence described above, but nevertheless the biological A mutant protein having one of the activities is also understood to mean specifically. Thus, a “functional equivalent” includes a mutated protein resulting from a mutation, where a particular change may occur at any sequence position, provided that the change has a profile of properties according to the present invention. To bring protein).

  “Mutation” for the purposes of the present invention means a change in the nucleic acid sequence of a genetic variant in a plasmid or in the genome of an organism. Mutations can occur, for example, as a result of errors during replication, or can be due to a mutation source. A large number of biological, chemical or physical sources of mutagenesis are known to those skilled in the art, but the rate of natural variation in the cellular genome of an organism is very low.

  Mutations include substitutions, insertions, deletions of one or more nucleic acid groups. Substitution is understood to mean the replacement of individual nucleobases, where base transfer (substitution of purine bases with purine bases or substitution of pyrimidine bases with pyrimidine bases) and base conversion (substitution of pyrimidine bases with purine bases ( Or vice versa).

  Additions or insertions are understood to mean the incorporation of additional nucleic acid groups into the DNA and can result in reading frame movement. In this type of reading frame movement, “in-frame” insertion / addition is distinguished from “out-frame” insertion. In the case of “in-frame” insertion / addition, the reading frame is maintained, resulting in a polypeptide that is increased by the number of amino acids encoded by the inserted nucleic acid. In the case of “out-frame” insertion / addition, the original reading frame is lost and the formation of a fully functional polypeptide is no longer possible.

  Deletions represent the loss of one or more base pairs, which also results in “in-frame” or “out-frame” reading frame shifts and associated results with respect to complete protein formation.

  Mutagens (mutagens) that can be used to generate random or target mutations and applicable methods and techniques are known to those skilled in the art. Such methods and mutagens are described, for example, by AM van Harten [(1998), “Mutation breeding: theory and practical applications”, Cambridge University Press, Cambridge, UK], E Friedberg, G Walker, W Siede [(1995) "DNA Repair and Mutagenesis", Blackwell Publishing], or K. Sankaranarayanan, JM Gentile, LR Ferguson [(2000) "Protocols in Mutagenesis", Elsevier Health Sciences].

  PCR mutagenesis using, for example, vitro mutagense kits, LA PCR in vitro Mutagenesis Kit (Takara Shuzo, Kyoto), or Stratagene QuikChange® Kit, or appropriate primers to introduce targeted mutations Conventional molecular biology methods and processes such as can be used.

  As already mentioned above, there are numerous chemical, physical and biological mutagens.

  The listed mutation sources are shown as examples below, but are not limiting.

  Chemical mutagens can be subdivided according to their mechanism of action. Base analogs (e.g., 5-bromouracil, 2-aminopurine), monofunctional and bifunctional alkylating agents (e.g., monofunctional ones such as ethyl methyl sulfonic acid, dimethyl sulfate, or dichlorosulfite dichloromethane) Bifunctional ones such as ethyl, mitomycin, nitrosoguanidine, dialkylnitrosamine, N-nitrosoguanidine derivatives), or intercalating substances (eg, acridine, ethidium bromide).

  Thus, for example, for the method according to the invention, obtained as a result of a mutation of a polypeptide according to the invention, for example SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 , 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124 , 126, 128, 130, 132, 134, 136, 138, 140, 146, 150, 153, 156, 157, 158, 160, 162, 164, 166, 168 and / or 170 may also be used. Is possible.

Examples of suitable amino acid substitutions are shown in the table below.

  In SEQ ID NO: 2, it is known that the serine naturally occurring at position 2849 can be substituted with, for example, glycine to avoid phosphorylation at this position (Fontao L, Favre B, Riou S , Geerts D, Jaunin F, Saurat JH, Green KJ, Sonnenberg A, Borradori L., Interaction of the bullous pemphigoid antigen 1 (BP230) and desmoplakin with intermediate filaments is mediated by distinct sequences within their COOH terminus., Mol Biol Cell. 2003 May; 14 (5): 1978-92. Epub 2003 Jan 26).

  In the above sense, “functional equivalents” are also “precursors” of the described polypeptides, as well as “functional derivatives” and “salts” of the polypeptides.

  As used herein, a “precursor” is a natural or synthetic precursor of a polypeptide, regardless of the presence or absence of a desired biological activity.

  The expression “salt” is understood to mean a salt of a carboxyl group or an acid addition salt of an amino group of the protein molecule of the invention. Salts of carboxyl groups can be prepared by methods known per se, for example, inorganic salts such as sodium, calcium, ammonium, iron and zinc salts; and also, for example, amines (such as triethylamine), arginine, lysine, piperidine And salts having an organic base such as Acid addition salts, such as salts with mineral acids such as hydrochloric acid or sulfuric acid, and salts with organic acids such as acetic acid and oxalic acid are also provided by the present invention.

  “Functional equivalents” naturally include polypeptides obtainable from other organisms, as well as naturally occurring variants (alleles). For example, the range of homologous sequence regions or conserved regions can be determined by sequence comparison. Using these sequences, DNA databases (eg, genomic or cDNA databases) can be examined for equivalent enzymes using bioinformatics comparison programs. Suitable computer programs and databases available to the public are well known to those skilled in the art.

  These alignments of known protein sequences can be performed, for example, using a computer program such as InforMax's vector NTI8 (September 25, 2002 edition).

  Furthermore, a “functional equivalent” has one of the above polypeptide sequences or functional equivalents derived therefrom and is functionally different from them in an N-terminal or C-terminal functional linkage (ie, a fusion). A fusion protein having at least one additional heterologous sequence (without mutual intrinsic dysfunction of the protein parts). Non-limiting examples of such heterologous sequences are, for example, signal peptides or enzymes.

  “Functional equivalents” encompassed by the present invention are homologues of the specifically disclosed proteins. When calculated using the computer programs and computer algorithms disclosed in the definitions, these are at least 40%, 45% or 50%, preferably at least 55%, especially for one of the disclosed amino acid sequences 60%, 65% or 70%, particularly preferably at least 75%, 80%, 85%, 90%, 91%, 92%, 93% or 94%, very particularly preferably at least 95% or 96% Have homology.

  Where protein glycosylation can occur, “functional equivalents” according to the present invention include deglycosylated or glycosylated forms, and also modified forms of the above types that can be obtained by altering glycosylation patterns. Contains protein.

  Where protein phosphorylation can occur, “functional equivalents” according to the present invention include dephosphorylated or phosphorylated forms, and also modified forms of the above types that can be obtained by altering phosphorylation patterns. Contains protein.

  A homologue of a polypeptide according to the invention can be identified by screening a combinatorial bank of variants, for example by shortening the variant. For example, a bank of protein variants can be generated by combinatorial mutagenesis at the nucleic acid level, for example, by enzymatic ligation of synthetic oligonucleotide mixtures. There are a number of methods that can be used to obtain a bank of homologue candidates from a degenerate oligonucleotide sequence. Chemical synthesis of degenerate gene sequences can be performed in an automated DNA synthesizer, and then the synthetic gene can be ligated into an appropriate expression vector. The use of a degenerate set of genes makes it possible to provide all the sequences that encode the desired set of protein sequence candidates in one mixture. Methods for synthesizing degenerate oligonucleotides are known to those skilled in the art (eg, Narang, SA (1983) Tetrahedron 39: 3; Itakura et al. (1984) Annu. Rev. Biochem. 53: 323; Itakura et al (1984) Science 198: 1056; Ike et al. (1983) Nucleic Acids Res. 11: 477).

  In the prior art, techniques are known for screening gene products in combinatorial banks generated by point mutations or truncations, and several techniques for screening cDNA banks for gene products with selected properties. . The most used technique for screening large gene banks subject to high-throughput analysis involves cloning the gene bank into a replicable expression vector and transforming appropriate cells with the resulting vector bank. Detection of the desired activity includes expressing the combinatorial gene under conditions that facilitate isolation of the vector encoding the gene from which the product has been detected. Recursive ensemble mutagenesis (REM), a technique that increases the frequency of functional variants in banks, can be used in combination with screening tests to identify homologs (Arkin and Yourvan (1992 ) PNAS 89: 7811-7815; Delgrave et al. (1993) Protein Engineering 6 (3): 327-331).

  SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 145, 149, 152, 159, 161, 163, 165, 167 and / or 169, particularly preferably using the nucleic acid sequence shown in SEQ ID NO: 165 and 167, most preferably the nucleic acid sequence shown in SEQ ID NO: 167, or a portion thereof as a probe. Examination of cDNA or genomic DNA libraries of other organisms that are publicly available are methods known to those skilled in the art for identifying homologues in other ways. Here, SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 145, 149, 152 , 159, 161, 163, 165, 167 and / or 169, particularly preferably 165 and 167, most preferably a probe derived from a nucleic acid sequence according to 167 is at least 20 bp, preferably at least 50 bp, particularly preferably at least 100 bp, Very particularly preferably it has a length of at least 200 bp, most preferably at least 400 bp. The probe can also have a length of 1 kilobase or more, for example, 1 Kb, 1.5 Kb or 3 Kb. To examine the library, SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, from 20 bp to several kilobases in length 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 145, 149, 152, 159, 161, 163, 165, 167 and / or 169, particularly preferably 165 and 167, most preferably 167 of the complementary DNA strand It may also be possible to use sequences, or fragments thereof. The hybridization conditions used are described above.

  In the method of the present invention, in a standard state, SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 145, 149, 152, 159, 161, 163, 165, 167 and / or 169, particularly preferably SEQ ID NO: 165 and 167, most preferably SEQ ID NO: 167 and a nucleic acid molecule encoding a keratin binding polypeptide DNA molecules that hybridize with and SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 as complete sequences , 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88 , 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 146, 150, 153, 156, 157, It is also possible to use DNA molecules that encode polypeptides having the same properties as the polypeptides shown in 158, 160, 162, 164, 166, 168 or 170.

  Particularly advantageous embodiments of the invention are SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, A keratin-binding polypeptide (ii) comprising at least one of the polypeptide sequences shown in 140, 146, 150, 153, 156, 157, 158, 160, 162, 164, 166, 168 or 170, provided that The keratin binding of the polypeptide, as measured in the test according to Example 9 or 10, is SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28. , 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78 , 80, 82, 84, 86, 88, 90, 92, 94, 96 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 146, 150, 153, At least 10%, 20%, 30%, 40% or 50%, preferably 60% of the value of the corresponding polypeptide sequence shown in 156, 157, 158, 160, 162, 164, 166, 168 or 170 70%, 80% or 90%, particularly preferably 100%.

  It is preferred to use a keratin-binding polypeptide (ii) that has a highly specific affinity for the desired organism. Therefore, for use in skin cosmetics, it is preferable to use a keratin-binding polypeptide (ii) having a particularly high affinity for human skin keratin. For use in cosmetics for hair, polypeptide sequences having a particularly high affinity for human hair keratin are preferred.

  For applications in the pet field, the described polypeptide sequences (SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 , 38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70,72,74,76,78,80,82,84,86 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136 , 138, 140, 146, 150, 153, 156, 157, 158, 160, 162, 164, 166, 168 or 170, preferably SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 40, 42 , 44, 46, 48, 146, 150, 153, 156, 157, 158, 160, 164, 166, 168 or 170, particularly preferably SEQ ID NO: 166 and 168, most preferably SEQ ID NO: 168), corresponding A keratin-binding polypeptide (ii) having a particularly high affinity for keratins such as canine keratin or cat keratin is therefore preferred.

  However, it is also possible to use a plurality of keratin-binding polypeptides (ii) coupled to an effector molecule (i) according to the invention, e.g. high binding affinity for human skin keratin. The keratin-binding polypeptide (ii) possessed can be combined with other keratin-binding polypeptides (ii) having a high affinity for keratin of human hair to bind to the effector molecule. It is also possible to use a chimeric polypeptide comprising two or more copies of the same (and different) keratin-binding polypeptide (ii) or its keratin-binding domain. In that way, for example, it was possible to achieve particularly effective keratin binding.

  Suitable keratin binding polypeptides (ii) are known. For example, desmoplakin and plectin have keratin-binding domains (Fontao L, Favre B, Riou S, Geerts D, Jaunin F, Saurat JH, Green KJ, Sonnenberg A, Borradori L., Interaction of the bullous pemphigoid antigen 1 (BP230 ) and desmoplakin with intermediate filaments is mediated by distinct sequences within their COOH terminus., Mol Biol Cell. 2003 May; 14 (5): 1978-92.Epub 2003 Jan 26; Hopkinson SB, Jones JC., The N-terminus of The transmembrane protein BP180 interacts with the N-terminal domain of BP230, thus mediating keratin cytoskeleton anchorage to the cell surface at the site of the hemidesmosome, Mol Biol Cell. 2000 Jan; 11 (1): 277-86).

  The keratin-binding polypeptide (i) of the present invention can also be easily separated again from keratin (if desired). To this end, it is possible, for example, to use a keratin-containing rinse, so that the keratin-binding polypeptide (i) detaches from the previous binding to keratin and is saturated with keratin from the rinse. Alternatively, a rinse containing a high content of detergent (eg, SDS) can also be washed off.

  The keratin-binding polypeptides (i) according to the invention have a further field of application in human cosmetics, in particular skin care, nail care and hair care, animal care, skin care and skin processing.

  Preferably, the keratin-binding polypeptide (ii) of the present invention is used for skin cosmetics and hair cosmetics. They allow for a long duration of action of the effector molecules that protect or protect at high concentrations.

  In a particularly preferred embodiment of the invention, a keratin-binding polypeptide is used that has binding affinity for keratin from human skin, hair or nails.

In a particularly preferred embodiment, the present invention provides
(i) the keratin-binding polypeptide used is SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 146, 150, 153, 156, 157, 158, 160, 162, 164, 166, 168 or 170, preferably SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 40, 42, Including one of the sequences shown in 44, 46, 48, 146, 150, 153, 156, 157, 158, 160, 162, 164, 166, 168 or 170, particularly preferably 166 and 168, most preferably 168 ,
(j) The linker molecule (iii) used is maleimidocaproic acid,
(k) The above method is provided wherein the effector molecule (i) is selected from the group consisting of pantothenic acid, panthenol, pantolactone, ester of panthenol, ether of panthenol, and cation derivatized panthenol.

  In one embodiment, the keratin-binding polypeptide (ii) used in step (a) of the method of the invention and the maleimidocaproic acid-panthenol effector molecule (iv) are used in equimolar amounts. Preferably, the molar ratio of keratin binding polypeptide (ii) to maleimidocaproic acid-panthenol effector molecule (iv) is 1: 1 to 1: 5, preferably 1: 1, 1: 1.1 or 1: 1.2. , Preferably 1: 1.3 or 1: 1.4, particularly preferably 1: 1.5 or 1: 1.6, very particularly preferably 1: 1.7 or 1: 1.8, most preferably 1: 1.9 or 1: 2. Larger ratios can be selected depending on the number of linking groups present therein and / or accessibility in naturally folded polypeptides on the surface. When using keratin binding domain B (SEQ ID NO: 166) it is also possible to choose a ratio of for example 1: 4, in which case this ratio is for example the hair binding activity of the keratin binding effector molecule formed. Affects (see Example 24).

  The invention further provides a keratin-binding effector molecule, wherein the effector molecule (i) is indirectly coupled to the keratin-binding polypeptide via a linker molecule (iii). SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 146, 150, 153, 156, 157 or 158, preferably SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 40, 42, 44, 46, 48, 146, 150, 153, 156, 157, 158, 160, 162, 164, A keratin-binding effector molecule comprising at least one keratin-binding polypeptide (ii) according to the sequence shown in 166, 168 or 170, particularly preferably 166 and 168, most preferably 168 is preferred and used during its production The linker molecule (iii) was maleimidocaproic acid. Particularly preferred keratin binding effector molecules are listed in Tables 12 and 12a. The linker molecule (iii) used is maleimidocaproic acid and the effector molecule (i) used is pantothenic acid, panthenol, pantolactone, an ester of panthenol, an ether of panthenol or a cation-derivatized panthenol. The above-mentioned keratin-binding effector molecule was very particularly preferred.

  The present invention further provides the use of the keratin-binding effector molecule produced according to the present invention in a skin cosmetic preparation. Preferably, the keratin-binding effector molecule of the present invention is used in skin and hair cosmetics. They allow long action times at high concentrations of skin care or skin protective effector substances. Furthermore, the use of keratin-binding effector molecules in gum and oral care is preferred.

  In a preferred embodiment of the invention, the keratin-binding effector molecule according to the invention and / or produced by the method of the invention is applied to skin cosmetics or oral, dental and denture care compositions to the total weight of the composition. 0.001 to 1% by weight (wt%), preferably 0.01 to 0.9% by weight, particularly preferably 0.01 to 0.8% by weight, or 0.01 to 0.7% by weight, very particularly preferably 0.01 to 0.6% by weight, or 0.01 It is added at a concentration of -0.5 wt%, most preferably 0.01-0.4 wt%, or 0.01-0.3 wt%. In a further embodiment, the composition comprises 1-10% by weight, preferably 2-8% by weight of keratin-binding effector molecule according to the invention and / or produced by the method of the invention, based on the total weight of the composition. %, 3-7% by weight, 4-6% by weight. In a likewise preferred embodiment, the composition comprises 10-20% by weight, preferably 11-11%, of keratin-binding effector molecules according to the invention and / or produced by the method of the invention, based on the total weight of the composition. Contains at concentrations of 19%, 12-18%, 13-17%, 14-16% by weight. In a more preferred embodiment, the composition comprises 20-30% by weight of keratin-binding effector molecules according to the invention and / or produced by the method of the invention, preferably 21-29%, based on the total weight of the composition. Containing in concentrations of wt%, 22-28 wt%, 23-27 wt%, 24-26 wt%.

  In another preferred embodiment, the keratin-binding effector molecule according to the invention as described above comprises (i) a cosmetic aid from the field of decorative cosmetics, (ii) a skin cosmetic active ingredient, and (iii) suitable auxiliaries and Used in combination with additives for skin cosmetics and / or oral, dental and denture care compositions. Preferably, they treat existing damage to the skin, hair, and / or fingernails or toenails to protect the skin, hair, and / or fingernails or toenails from damage Active ingredients and auxiliaries and additives that are used to do and to care for the skin, hair, and / or fingernails or toenails. These active ingredients are preferably from the group of natural or synthetic polymers, pigments, wetting agents, oils, waxes, enzymes, minerals, vitamins, sunscreens, dyes, fragrances, antioxidants, preservatives and / or pharmaceutically active ingredients. Is selected.

  Suitable auxiliaries and additives for producing hair cosmetic or skin cosmetic preparations are well known to those skilled in the art and are described in cosmetic handbooks such as Schrader, Grundlagen und Rezepturen der Kosmetika [Fundamentals and formulations of cosmetics]. , Huthig Verlag, Heidelberg, 1989, ISBN 3-7785-1491-1, or Umbach, Kosmetik: Entwicklung, Herstellung und Anwendung kosmetischer Mittel [Cosmetics: development, manufacture and use of cosmetic compositions], 2nd expanded edition, 1995, Georg Thieme Can be found in Verlag, ISBN 3 13 712602 9.

  Preferably, the keratin-binding effector molecule of the present invention is used in skin cosmetics or oral care, dental care and denture care compositions in cosmetic active ingredients, emulsifiers, surfactants, preservatives, perfume oils. , Thickener, hair polymer, hair conditioner and skin conditioner, graft polymer, water-soluble or dispersible silicone-containing polymer, photoprotective agent, bleaching agent, gel-forming agent, care agent, coloring agent, coloring agent, tanning agent, pigment At least one different from that selected from, pigments, viscosity modifiers, wetting agents, fat additives, collagen, protein hydrolysates, lipids, antioxidants, antifoams, antistatic agents, emollients and emollients Used in combination with a variety of ingredients. The active ingredient is encapsulated in a cosmetic preparation as described in patents / patent applications EP00974775B1, DE2311712, EP0278878, DE199947147, EP0706822B1 and WO98 / 16621, which are expressly incorporated herein by reference. It can also exist in a form.

Conveniently the antioxidant is an amino acid (e.g. glycine, histidine, tyrosine, tryptophan) and derivatives thereof, imidazole (e.g. urocanic acid) and derivatives thereof, peptides such as D, L-carnosine, D-carnosine, L -Carnosine and its derivatives (e.g. anserine), carotenoids, carotenes (e.g. β-carotene, lycopene) and its derivatives, chlorogenic acid and its derivatives, lipoic acid and its derivatives (e.g. dihydrolipoic acid), aurothioglucose, propyl Thiouracil and other thiols (e.g. thioroxine, glutathione, cysteine, cystine, cystamine and their glycosyl, N-acetyl, methyl, ethyl, propyl, amyl, butyl and lauryl, palmitoyl, oleyl, γ-linoleyl, core Teryl and glyceryl esters) and their salts, dilauryl thiodipropionate, distearyl thiodipropionate, thiodipropionic acid and its derivatives (esters, ethers, peptides, lipids, nucleotides, nucleosides and salts), and sulfoximine compounds ( For example, very low tolerances (e.g., pmol to μmol / kg) of buthionine sulphoximine, homocysteine sulphoximine, buthionine sulphone, pentathionine sulphoximine, hexathionine sulphoximine, heptathionine sulphoximine ), As well as (metal) chelating agents (e.g. α-hydroxy fatty acids, palmitic acid, phytic acid, lactoferrin), α-hydroxy acids (e.g. citric acid, lactic acid, malic acid), humic acid, bile acids, bile extracts , Bilirubin, biliverdin EDTA and derivatives thereof, unsaturated fatty acids and derivatives thereof (e.g. γ-linolenic acid, linoleic acid, oleic acid), folic acid and derivatives thereof, ubiquinone and ubiquinol and derivatives thereof, vitamin C and derivatives thereof (e.g. sodium ascorbate, Ascorbyl palmitate, magnesium ascorbyl phosphate, ascorbyl acetate), tocopherols and derivatives (e.g. vitamin E acetate, tocotrienol), vitamin A and derivatives (vitamin A palmitate), and benzoin coniferyl benzoate, rutinic acid and its derivatives, α-Glycosyl rutin, ferulic acid, furfurylidene glucitol, carnosine, butylhydroxytoluene, butylhydroxyanisole, nordihydroguaiacic acid, nordihi De log Aia retinoic acid (Nordihydroguaiaretic acid), tri-hydroxybutyronitrile phenone, uric acid and derivatives thereof, mannose and derivatives thereof, zinc and derivatives thereof (e.g., ZnO, ZnSO _4), selenium and derivatives thereof (e.g., selenomethionine), stilbenes and It is selected from the group consisting of its derivatives (eg stilbene oxide, trans-stilbene oxide).

  Examples of vitamin B group vitamins, provitamins or vitamin precursors, or derivatives thereof, and derivatives of 2-furanone that are preferably used according to the present invention include:

Vitamin B ₁ , common name thiamine, chemical name 3-[(4′-amino-2′-methyl-5′-pyrimidinyl) methyl] -5- (2-hydroxyethyl) -4-methylthiazolium chloride.

Vitamin B ₂ , common name riboflavin, chemical name 7,8-dimethyl-10- (1-D-ribityl) -benzo [g] pteridine-2,4 (3H, 10H) -dione. In free form, riboflavin is present in free form, for example in whey, and other riboflavin derivatives can be isolated from bacteria and yeast. A riboflavin stereoisomer that is also suitable in the present invention is roxoflavin, which can be isolated from fish meal or liver and has a D-arabityl group instead of a D-ribityl group.

Vitamin B ₃ . The compounds of nicotinic acid and nicotinamide (niacinamide) often have this name. According to the invention, nicotinamide is preferred.

Vitamin B ₅ (pantothenic acid and panthenol). It is preferred to use panthenol. It is preferred to use panthenol. Panthenol derivatives that can be used according to the invention are in particular esters and ethers of panthenol and cation-derivatized panthenol. In a further preferred embodiment of the invention, in addition to pantothenic acid or panthenol, derivatives of 2-furanone can also be used. Particularly preferred derivatives are also the commercially available substances dihydro-3-hydroxy-4,4-dimethyl-2 (3H) -furanone (Merck), 4-hydroxymethyl-γ, the common name pantolactone. -Butyrolactone (Merck), 3,3-dimethyl-2-hydroxy-γ-butyrolactone (Aldrich) and 2,5-dihydro-5-methoxy-2-furanone (Merck). Clearly includes all stereoisomers.

  Advantageously, these compounds impart moisturizing and skin soothing properties to the skin cosmetics of the present invention.

Vitamin B ₆ . This is not a uniform substance, but a derivative of 5-hydroxymethyl-2-methylpyridin-3-ol known by the common names pyridoxine, pyridoxamine and pyridoxal.

Vitamin B ₇ (biotin) is also referred to as vitamin H or “skin vitamin”. Biotin is (3aS, 4S, 6aR) -2-oxohexahydrothienol [3,4-d] imidazole-4-valeric acid.

  Panthenol, pantolactone, nicotinamide and biotin are very particularly preferred according to the invention.

Dyes that can be used are listed, for example, in the publication “Kosmetische Farbemittel” [Cosmetic Colorants] (published by Verlag Chemie, Weinheim) 1984 from Farbstoffkommission der Deutschen Forschungsgemeinschaft [Dyes Commission of the German Research Society]. As such, it is a suitable substance approved for cosmetic use. These dyes are usually used at a concentration of 0.001 to 0.1% by weight based on the total mixture.

Pigment In a preferred embodiment, the composition according to the invention comprises at least one pigment. The pigment is present in undissolved form in the product mass and may be present in an amount of 0.01 to 25% by weight, particularly preferably 5 to 15% by weight. The preferred particle size is 1 to 200 μm, in particular 3 to 150 μm, particularly preferably 10 to 100 μm. The pigment is a colorant that is substantially insoluble in the coating medium and may be inorganic or organic. Inorganic and organic mixed pigments are also possible. Inorganic pigments are preferred. The advantage of inorganic pigments is their excellent light stability, weather stability and thermal stability. Inorganic pigments may be of natural origin prepared from, for example, chalk, ocher, amber, green clay, burnt schener or graphite. Pigments include white pigments such as titanium dioxide or zinc oxide; black pigments such as black iron oxide; colored pigments such as ultramarine or red iron oxide; pearlescent pigments; metal effect pigments; pearlescent pigments and fluorescent pigments or The luminescent pigment may be used, and the at least one pigment is preferably a colored non-white pigment. Metal oxides, metal hydroxides and metal oxide hydrates, mixed phase pigments, sulfur-containing silicates, metal sulfides, complex metal cyanides, metal sulfates, metal chromates, metal molybdates, And the metal itself (bronze pigment) is suitable. Titanium dioxide (CI77891), black iron oxide (CI77499), yellow iron oxide (CI77492), red and brown iron oxide (CI77491), manganese violet (CI77742), ultramarine (sodium aluminum sulfosilicate, CI77007, pigment blue 29), Particularly suitable are chromium oxide hydrate (CI77289), bitumen (ferric blue ferrocyanide, CI77510) and carmine (cochineal). Pearls based on mica coated with metal oxides or metal oxychlorides such as titanium dioxide or bismuth oxychloride and, where appropriate, further color imparting materials such as iron oxide, bitumen, ultramarine, carmine, etc. Glossy pigments and colored pigments are particularly preferred, and the color can be determined by changing the thickness of the layer. This type of pigment is sold, for example, under the trade names Rona®, Colorona®, Dichrona® and Timiron® (Merck). Organic pigments are, for example, the natural pigments sepia, gunborg, kassel brown, indigo, chlorophyll and other plant pigments. Synthetic organic pigments are, for example, azo pigments, anthraquinoids, indigoids, dioxazines, quinacridones, phthalocyanines, isoindolinones, perylenes and perinones, metal complexes, alkali blue and diketopyrrolopyrrole pigments.

  In one embodiment, the keratin-binding effector molecule according to the invention and / or produced by the method of the invention is at least one kind present in the composition in an amount of 0.01 to 10% by weight, preferably 0.05 to 5% by weight. Used with other particulate matter. Suitable substances are, for example, substances which are solid at room temperature (25 ° C.) and in the form of particles. For example, silica, silicates, aluminates, clay soils, mica, salts, especially inorganic metal salts, metal oxides such as titanium dioxide, minerals and polymer particles are suitable. The particles are not dissolved in the composition and are preferably present in a stably dispersed form and can be applied in solid form after application to the application surface and evaporation of the solvent. Preferred particulate materials are silica (silica gel, silicon dioxide) and metal salts, especially inorganic metal salts, with silica being particularly preferred. The metal salt is, for example, an alkali metal halide or alkaline earth metal halide such as sodium chloride or potassium chloride; an alkali metal sulfate or alkaline earth metal sulfate such as sodium sulfate or magnesium sulfate.

Pearlescent brighteners Suitable pearlescent brighteners are, for example, alkylene glycol esters, in particular ethylene glycol distearate; fatty acid alkanolamides, in particular coconut oil fatty acid diethanolamide; partial glycerides, in particular stearic acid monoglycerides; optionally hydroxy-substituted Esters of polybasic carboxylic acids with fatty alcohols having 6 to 22 carbon atoms, in particular long-chain esters of tartaric acid; for example having at least 24 carbon atoms in total, for example fatty alcohols, fatty ketones, fatty aldehydes, Fatty substances such as fatty ethers and fatty carbonates, especially laurone and distearyl ethers; fatty acids such as stearic acid, hydroxystearic acid or behenic acid; olefin epoxides having 12 to 22 carbon atoms and 12 to 22 carbon atoms Having fat al Ring-opening products with coal and / or with polyols having 2 to 15 carbon atoms and 2 to 10 hydroxyl groups, and mixtures thereof.

  Conventional thickeners in such formulations are cross-linked polyacrylic acid and derivatives thereof, polysaccharides and derivatives thereof such as xanthan gum, agar, alginate or tyrose, cellulose derivatives such as carboxymethylcellulose or hydroxycarboxymethylcellulose. Fatty alcohols, monoglycerides and fatty acids, polyvinyl alcohol and polyvinyl pyrrolidone. It is preferred to use a nonionic thickener.

  Suitable cosmetic and / or skin cosmetic active ingredients are, for example, coloring active ingredients, skin and hair dyes, coloring agents, tanning agents, bleaching agents, keratin-curing substances, antibacterial active ingredients, light filter active ingredients, Repellent active ingredients, hyperemic substances, substances with keratolytic and keratin softening effects, anti-dandruff active ingredients, anti-inflammatory agents, keratinizing substances, antioxidant active ingredients and / or active ingredients that act as free radical scavengers, Skin moisturizing or moisturizing substances, fat active ingredients, anti-erythemal or anti-allergic active ingredients, branched fatty acids such as 18-methyl icosanoic acid, and mixtures thereof.

  Artificial skin tanning active ingredients suitable for browning the skin without natural or artificial irradiation with ultraviolet light are, for example, dihydroxyacetone, alloxan and walnut shell extracts. Suitable keratin-curing substances are active ingredients such as, for example, potassium aluminum sulfate, aluminum hydroxychloride, aluminum lactate, which are also commonly used in antiperspirants.

  Antibacterial active ingredients are used to extinguish microorganisms or inhibit their growth and thus serve both as preservatives and as deodorants that reduce the occurrence or increase of body odor. These include, for example, conventional preservatives known to those skilled in the art such as p-hydroxybenzoic acid ester, imidazolidinyl urea, formaldehyde, sorbic acid, benzoic acid, salicylic acid and the like. Such deodorizing substances are, for example, zinc ricinoleate, triclosan, undecylenic acid alkylolamides, triethyl citrate, chlorhexidine and the like.

Suitable preservatives advantageously used according to the present invention are as follows:

  Also suitable according to the invention are dibromodicyanobutane (2-bromo-2-bromomethylglutaronitrile), a preservative or preservative aid customary in cosmetics, 3-iodo-2-propynyl butylcarbamate, 2-Bromo-2-nitropropane-1,3-diol, imidazolidinyl urea, 5-chloro-2-methyl-4-isothiazolin-3-one, 2-chloroacetamide, benzalkonium chloride and benzyl alcohol. Also suitable as preservatives are phenylhydroxyalkyl ethers, in particular the compounds known under the name phenoxyethanol for their bactericidal and fungicidal effects against several microorganisms.

  Other antimicrobial agents are equally suitable for incorporation into the preparations according to the invention. Advantageous substances are, for example, 2,4,4′-trichloro-2′-hydroxydiphenyl ether (Irgasan), 1,6-di (4-chlorophenylbiguanide) hexane (chlorhexidine), 3,4,4′-trichlorocarbohydrate. Anilides, quaternary ammonium compounds, clove oil, peppermint oil, thyme oil, triethyl citrate, farnesol (3,7,11-trimethyl-2,6,10-dodecatrien-1-ol), and patent specification DE-3740186, DE-3938140, DE-4204321, DE-4229707, DE-4309372, DE-4411664, DE-19541967, DE-19543695, DE-19543696, DE-19547160, DE-19602108, DE-19602110, DE- 19602111, DE-19631003, DE-19631004 and DE-19634019 and the active ingredients or active ingredients described in the patent specifications DE4229737, DE-4237081, DE-4324219, DE-4429467, DE-4423410 and DE-19516705 It is a combination. Sodium bicarbonate is also conveniently used. Microbial polypeptides can be used as well.

The perfume cosmetic composition may comprise perfume oil where appropriate. Perfume oils that can be mentioned are, for example, mixtures of natural and synthetic fragrances. Natural flavors include flowers (lily, lavender, rose, jasmine, neroli, ylang ylang), stems and leaves (geranium, patchouli, petit glen), fruits (aniseed, coriander, caraway, juniper), peel (bergamot, lemon, orange ), Root (mace, angelica, celery, cardamom, costas, iris, agate), tree (pine, sandalwood, guayakwood, cedarwood, rosewood), herb and grass (taragon, lemongrass, sage, thyme), conifer Extracted from rosewood, herbs and grass (tarragon, lemongrass, sage, thyme), needles and branches (spruce, fir, pine, shrub pine), resin and balsam (galvanum, elemi, benzoin, myrrh, olivanum, opoponax) The Also suitable are animal raw materials such as, for example, civet and beaver incense. Typical synthetic perfume compounds are esters, ethers, aldehydes, ketones, alcohols and hydrocarbon products. Ester-type fragrance compounds include, for example, benzyl acetate, phenoxyethyl isobutyrate, 4-tert-butylcyclohexyl acetate, linalyl acetate, dimethylbenzylcarbyl acetate, phenylethyl acetate, linalyl benzoate, benzyl formate, ethyl methyl phenyl glycinate , Allyl cyclohexylpropionate, styrylyl propionate and benzyl salicylate. Ethers include, for example, benzyl ethyl ether, and aldehydes include, for example, alkanals having 8 to 18 carbon atoms, citral, citronellal, citronellyloxyacetaldehyde, cyclamenaldehyde, hydroxycitronellal, lyial and Examples include burgeonals; ketones include, for example, ionone, isomethylionone, and methylcedryl ketone; alcohols include anethole, citronellol, eugenol, isoeugenol, geraniol, linalool, phenylethyl alcohol, and terpeneol. The hydrocarbons mainly include terpenes and balsams. However, it is preferred to use a mixture of different fragrances that together produce a pleasant scent. Relatively low volatility essential oils that are mainly used as aromatic components are also suitable as perfume oils, for example, sage oil, chamomile oil, clove oil, melissa oil, mint oil, cinnamon oil, linden flower oil, pine matsuko Oil, vetiver oil, olibanum oil, galvanum oil, radinam oil and lavender oil. Preferably, bergamot oil, dihydromyrsenol, lyral, rilal, citronellol, phenylethyl alcohol, α-hexylcinnamaldehyde, geraniol, benzylacetone, cyclamenaldehyde, linalool, Boisambrene® Forte, ambroxan, indole, hedione (hedione), Sandelis, lemon oil, mandarin oil, orange oil, allyl amyl glycolate, cyclovertal, lavandin oil, clarisage oil, β-damascon, geranium oil bourbon, cyclohexyl salicylate, Vertofix® Coeur, Iso- E-Super®, Fixolide® NP, evanil, iraldein gamma, phenylacetic acid, geranyl acetate, benzyl acetate, rose oxide, romillate, irotyl, and furoyl Floramate is used alone or in a mixture.

Oils, fats and waxes Preferably, the composition according to the invention comprises oils, fats and / or waxes. The components of the oil phase and / or fatty phase of the composition according to the invention are lecithin and fatty acid triglycerides, i.e. saturated and / or unsaturated branches having a chain length of 8 to 24, in particular 12 to 18 carbon atoms. Are advantageously selected from the group of triglycerol esters of alkanecarboxylic acids which are in the form of and / or unbranched. Fatty acid triglycerides are, for example, synthetic, semi-synthetic and natural oils such as olive oil, sunflower oil, soybean oil, peanut oil, rapeseed oil, tonsil oil, palm oil, coconut oil, castor oil, wheat germ oil, grape seed oil , And can be conveniently selected from the group of thistle oil, evening primrose oil, macadamia nut oil and the like. The further polar oil component comprises a saturated and / or unsaturated branched and / or unbranched alkanecarboxylic acid having a chain length of 3 to 30 carbon atoms and a chain length of 3 to 30 carbon atoms. From saturated and / or unsaturated branched and / or unbranched esters with esters, and aromatic carboxylic acids and saturated and / or unsaturated having a chain length of 3 to 30 carbon atoms From the group of esters with branched and / or unbranched alcohols. And such ester oils include isopropyl myristate, isopropyl palmitate, isopropyl stearate, isopropyl oleate, n-butyl stearate, n-hexyl laurate, n-decyl oleate, isooctyl stearate, isononyl stearate, Isononyl isononanoate, 2-ethylhexyl palmitate, 2-ethylhexyl laurate, 2-hexyldecyl stearate, 2-octyldodecyl palmitate, oleyl oleate, oleyl erucate, erucyl oleate, dicaprylyl erucyl carbonate (cetiol CC ) And cocoglycerides (myritol 331), dicaprylic acid / butylene glycol dicaprate and dibutyl adipate, and synthetic, semi-synthetic and natural mixtures (such as jojoba oil) of such esters It can be conveniently selected from the group.

  Furthermore, the one or more oily components are advantageously from the group of branched and unbranched hydrocarbons and hydrocarbon waxes, silicone oils, dialkyl ethers, saturated or unsaturated branched or unbranched alcohols. You can choose. Any mixture of such oil and wax components can also be conveniently used for the purposes of the present invention. Where appropriate, it may also be advantageous to use a wax, for example cetyl palmitate, as the sole lipid component of the oil phase. According to the present invention, the oily components are 2-ethylhexyl isostearate, octyldodecanol, isotridecyl isononanoate, isoeicosane, coconut oil fatty acid 2-ethylhexyl, alkyl C12-15-benzoate, caprylic / capric triglyceride, dicapryl Conveniently selected from the group consisting of ruether. According to the invention, a mixture of C12-15-alkyl benzoate and 2-ethylhexyl isostearate, a mixture of C12-15-alkyl benzoate and isotridecyl isononanoate, and C12-15-alkyl benzoate and isostearic acid 2 Preference is given to a mixture of ethylhexyl and isotridecyl isononanoate. According to the invention, oils having a polarity of 5 to 50 mN / m that are particularly preferably used are fatty acid triglycerides, in particular soybean oil and / or tonsil oil. In hydrocarbons, paraffin oil, squalane and squalene are conveniently used for the purposes of the present invention.

Furthermore, the oil phase can be conveniently selected from the group of Gerve alcohol. Gerve alcohol is named after Marcel Guerbet, who first described its preparation. They are reaction equations:

Aldehyde is formed by oxidation of alcohol, and water is removed from the aldol by the aldol condensation of the aldehyde and allylaldehyde is hydrogenated. Gerve alcohol is liquid even at low temperatures and has virtually no skin irritation effect. They can be conveniently used as fat, overfat and also fat ingredients in cosmetic compositions.

The use of Gerve alcohol in cosmetics is known per se. Such species are then mostly structured:

Is characterized by Here, R ₁ and R ₂ are usually unbranched alkyl groups.

According to the present invention, Gerve alcohol or alcohol is
R ₁ = propyl, butyl, pentyl, hexyl, heptyl or octyl, and
R ₂ = conveniently selected from the group of hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl or tetradecyl.

  Gerve alcohols preferred according to the present invention are 2-butyl octanol (e.g. commercially available as Isofol® 12 (Condea)) and 2-hexyldecanol (e.g. Isofol® 16 (Condea)). It is. Mixtures of Gerve alcohol according to the invention, such as, for example, a mixture of 2-butyloctanol and 2-hexyldecanol (for example, commercially available as Isofol® 14 (Condea)) are also advantageously used according to the invention. The

  Any mixture of such oil and wax components is also conveniently used for the purposes of the present invention. Of the polyolefins, polydecene is the preferred material.

The oily component can also conveniently have a component of a cyclic or linear silicone oil, or it can be composed entirely of such oils, but with silicone oils or several types of silicone oils Apart from that, it is preferred to use further components of other oil phase components. Low molecular weight silicones or silicone oils are generally defined by the following general formula:

Higher molecular weight silicones or silicone oils are generally defined by the following general formula:

In the formula, the silicon atom may be substituted here by the same or different alkyl and / or aryl groups, indicated here by their generic names by the radicals R _{1 to} R ₄ . However, the number of different groups is not necessarily limited to 4. Here, m can be a value between 2 and 200,000.

Cyclic silicones conveniently used according to the present invention are generally defined by the general formula:

In which the silicon atom may be substituted here by the same or different alkyl and / or aryl groups, indicated here in general terms by the radicals R _{1 to} R ₄ . However, the number of different groups is not necessarily limited to 4. Here, “n” can be a value of 3/2 to 20. The fractional value of n allows for a non-uniform number of siloxyl groups to be present in the ring.

  Conveniently, phenyltrimethicone is selected as the silicone oil. Other silicone oils such as dimethicone, hexamethylcyclotrisiloxane, phenyl dimethicone, cyclomethicone (octamethylcyclotetrasiloxane), hexamethylcyclotrisiloxane, polydimethylsiloxane, poly (methylphenylsiloxane), cetyl dimethicone, beheno Xidimethicone can also be conveniently used for the purposes of the present invention. Also advantageous are mixtures of cyclomethicone and isotridecyl isononanoate and of cyclomethicone and 2-ethylhexyl isostearate. However, it is also advantageous to select silicone oils whose composition is similar to the above-mentioned compounds whose organic side chains are derivatised, for example polyethoxylated and / or polypropoxylated. These include, for example, polysiloxane-polyalkyl-polyether copolymers, such as cetyl dimethicone copolyol. Cyclomethicone (octamethylcyclotetrasiloxane) is conveniently used as the silicone oil used according to the present invention. The fat and / or wax component that is advantageously used according to the invention can be selected from the group of vegetable waxes, animal waxes, mineral waxes and petrochemical waxes. For example, candelilla wax, carnauba wax, wax, agaric wax, cork wax, guarma wax, rice bran oil wax, sugar cane wax, berry wax, aurikuri wax, montan wax, jojoba wax, shea butter, beeswax Preference is given to shellac wax, whale wax, lanolin (wool wax), tail fat, ceresin, ozokerite (smellar wax), paraffin wax and microwax.

Further advantageous fat and / or wax components include chemically modified waxes and synthetic waxes such as Syncrowax® HRC (glyceryl tribehenate), Syncrowax® AW1C (C _18-36 fatty acids); montan ester waxes ; Sasol (sasol) waxes; hydrogenated jojoba waxes, synthetic or modified beeswax (e.g., dimethicone copolyol beeswax and / or C _{30 to 50} - alkyl beeswax); cetyl ricinoleate, e.g., Tegosoft (registered trademark) CR Polyalkylene waxes; polyethylene glycol waxes, and also chemically modified fats such as hydrogenated vegetable oils (e.g., hydrogenated castor oil and / or hydrogenated coconut fatty glycerides); triglycerides such as hydrogenated soy fatty acid glyceryl; trihydroxystearin; fatty acids; Fatty acid esters and glycol esters such as steers Phosphate C _{20 to 40} - alkyl, stearic acid C _{20 to 40} - alkyl hydroxy stearoyl, and / or glycol montanate. In addition, certain organosilicon compounds having physical properties similar to certain fat and / or wax components, such as stearoxytrimethylsilane, are also advantageous.

According to the invention, the fat and / or wax component can be used alone or as a mixture in the composition. Any mixture of such oil and wax components is also conveniently used for the purposes of the present invention. Conveniently, the oil phase comprises 2-ethylhexyl isostearate, octyldodecanol, isotridecyl isononanoate, dicaprylic acid / butylene glycol dicaprate, coconut oil fatty acid 2-ethylhexyl, C _12-15 -alkyl benzoate, caprylic acid / caprin It is selected from the group consisting of acid triglycerides and dicaprylyl ether. Mixture of octyldodecanol and caprylic / capric triglyceride, dicaprylyl ether, dicaprylyl carbonate and cocoglyceride, or a mixture of C _12-15 -alkyl benzoate and 2-ethylhexyl isostearate, C _12-15 -benzoic acid mixture of alkyl and butylene glycol dicaprylate / dicaprate, and C _{12 to 15} - a mixture of benzoic acid alkyl and isotridecyl, 2-ethylhexyl isostearate and isononanoate are particularly advantageous. For hydrocarbons, paraffin oil, cycloparaffin, squalane, squalene, hydrogenated polyisobutene and polydecene should be conveniently used for the purposes of the present invention.

The oily component is also conveniently selected from the group of phospholipids. Phospholipids are phosphate esters of acylated glycerol. The most important phosphatidylcholines are, for example, the general structure:

Wherein R ′ and R ″ are typically lecithins which are unbranched aliphatic groups having 15 or 17 carbon atoms and up to 4 cis double bonds.

  According to the present invention, Merkur Weissoel Pharma 40 from Merkur Vaseline, Shell Ondina (registered trademark) 917 from Shell & DEA Oil, Shell Ondina (registered trademark) 927, Shell Oil 4422, Shell Ondina (registered trademark) 933, Pionier (registered) Trademark) 6301S, Pionier® 2071 (Hansen & Rosenthal) can be conveniently used according to the present invention as paraffin oil. Suitable cosmetically compatible fats and oils are described in Karl-Heinz Schrader, Grundlagen und Rezepturen der Kosmetika [Fundamentals and formulations of cosmetics], 2nd edition, Verlag Huthig, Heidelberg, pp. It is described in 319-355.

Solvents if the keratin-binding effector molecules according to the invention and / or produced by the method of the invention are used in cosmetic or dermatological preparations which are solutions or emulsions or dispersions Solvents that can be used are water or aqueous solutions; oils such as capric acid or caprylic acid triglycerides, but preferably castor oil; fats, waxes and other natural and synthetic fatty substances, preferably fatty acids and low carbon number Esters with alcohols such as isopropanol, propylene glycol or glycerol or fatty alcohols with low alkanoic acids or with fatty acids; low carbon alcohols, diols or polyols and their ethers, preferably Ethanol, isopropano , Propylene glycol, glycerol, ethylene glycol, ethylene glycol monoethyl or monobutyl ether, propylene glycol monomethyl, monoethyl or monobutyl ether, diethylene glycol monomethyl or monoethyl ether and similar products. In particular, mixtures of the above solvents are used. In the case of alcoholic solvents, water may be a further component.

Surfactants According to the present invention, in addition to keratin-binding effector molecules according to the present invention and / or produced by the methods of the present invention, the composition can also include a surfactant. Such surfactants are, for example,
Phosphate esters and salts, such as DEA-oleth-10 phosphate and dilaureth-4 phosphate,
Alkyl sulfonates such as sodium coconut monoglyceride sulfate, sodium C _12-14 olefin sulfonate, sodium lauryl sulfoacetate and magnesium PEG-3 cocamide sulfate
Carboxylic acids and derivatives such as lauric acid, aluminum stearate, magnesium alkanolates and zinc undecylenate, ester carboxylic acids such as calcium stearoyl lactate, laureth-6 citrate and sodium PEG-4 lauramide carboxylate, etc.
Ester ethers formed by esterification of carboxylic acids with ethylene oxide, glycerol, sorbitan or other alcohols, such as ethoxylated alcohols, ethoxylated lanolins, ethoxylated polysiloxanes, propoxylated POE ethers and alkyl polyglycosides ( Lauryl glucoside, decyl glucoside and cocoglycoside).

Polysorbate According to the present invention, in addition to the keratin-binding effector molecule according to the present invention and / or produced by the method of the present invention, the composition may also comprise a polysorbate.

Here, polysorbates which are advantageous for the purposes of the present invention are:
・ Polyoxyethylene (20) sorbitan monolaurate (Tween 20, CAS No. 9005-64-5)
・ Polyoxyethylene (4) sorbitan monolaurate (Tween 21, CAS No. 9005-64-5)
・ Polyoxyethylene (4) sorbitan monostearate (Tween61, CAS No. 9005-67-8)
・ Polyoxyethylene (20) sorbitan tristearate (Tween65, CAS No. 9005-71-4)
・ Polyoxyethylene (20) sorbitan monooleate (Tween 80, CAS No. 9005-65-6)
・ Polyoxyethylene (5) sorbitan monooleate (Tween81, CAS No. 9005-65-5)
Polyoxyethylene (20) sorbitan trioleate (Tween 85, CAS number 9005-70-3).

Especially advantageous ones are
・ Polyoxyethylene (20) sorbitan monopalmitate (Tween 40, CAS No. 9005-66-7)
Polyoxyethylene (20) sorbitan monostearate (Tween 60, CAS number 9005-67-8).

  According to the invention, they are advantageously used individually or as a mixture of two or more polysorbates, at a concentration of 0.1-5% by weight, in particular at a concentration of 1.5-2.5% by weight, based on the total weight of the composition Is done.

Conditioning Agent In a preferred embodiment of the present invention, the composition also includes a conditioning agent. Conditioning agents preferred according to the invention are described, for example, in Section 4 of the International Cosmetic Ingredient Dictionary and Handbook (Volume 4, editor: RC Pepe, JA Wenninger, GN McEwen, The Cosmetic, Toiletry, and Fragrance Association, 9th edition, 2002). , Hair conditioning agent, wetting agent, skin conditioning agent, skin conditioning agent (softening agent), skin conditioning agent (moisturizing agent), skin conditioning agent (other), skin conditioning agent (occlusion), and skin protection agent All compounds listed, and all compounds listed as “water-soluble conditioning agents” and “oil-soluble conditioning agents” in EP-A934956 (pages 11-13). Further convenient conditioning agents are, for example, the compounds indicated by INCI as Polyquaternium (especially Polyquaternium-1 to Polyquaternium-56).

  Suitable conditioning agents also include, for example, polymeric quaternary ammonium compounds, cationic cellulose derivatives and polysaccharides.

Conditioning agents advantageous according to the invention can now be selected from the compounds shown in the table below:

  Further conditioners advantageous according to the invention are cellulose derivatives and quaternized guar gum derivatives, in particular guar hydroxypropylammonium chloride (e.g. Jaguar Excel®, Jaguar C162® (Rhodia), CAS65497-29- 2, CAS39421-75-5).

  Nonionic poly-N-vinylpyrrolidone / polyvinyl acetate copolymer (e.g. Luviskol® VA64 (BASF Aktiengesellschaft)), anionic acrylic acid copolymer (e.g. Luviflex® Soft (BASF Aktiengesellschaft) And / or amphoteric amide / acrylic acid / methacrylic acid copolymers (eg, Amphomer® (National Starch)) can be conveniently used in the present invention as conditioners.

Adding the powder raw material It may generally be convenient to add the powder raw material. The use of talc is particularly preferred.

Ethoxylated glycerol fatty acid esters According to the invention, in addition to the keratin-binding effector molecules produced according to the invention and / or by the method of the invention, the composition may, where appropriate, be an ethoxylated glycerol fatty acid ester, particularly preferably PEG-10 olive oil glyceride, PEG-11 avocado oil glyceride, PEG-11 cocoa butter glyceride, PEG-13 sunflower oil glyceride, PEG-15 glyceryl isostearate, PEG-9 coconut fatty acid glyceride, PEG-54 hydrogenated castor oil, PEG- 7 hydrogenated castor oil, PEG-60 hydrogenated castor oil, jojoba oil ethoxylate (PEG-26 jojoba fatty acid, PEG-26 jojoba alcohol), coconut oil fatty acid glyceres-5, PEG-9 coconut fatty acid glyceride, coconut oil fatty acid PEG-7 Glyceres, PEG-45 palm kernel oil glyceride, PEG-35 castor oil, olive oil PEG-7 ester, PEG-6 caprylic acid / capri Acid glyceride, PEG-10 olive oil glyceride, PEG-13 sunflower oil glyceride, PEG-7 hydrogenated castor oil, hydrogenated palm kernel oil glyceride PEG-6 ester, PEG-20 corn oil glyceride, (oleic acid palm fatty acid) PEG-18 Glyceryl, PEG-40 hydrogenated castor oil, PEG-40 castor oil, PEG-60 hydrogenated castor oil, PEG-60 corn oil glyceride, PEG-54 hydrogenated castor oil, PEG-45 palm kernel oil glyceride, PEG-35 castor oil , An ethoxylated oil selected from the group of coconut oil fatty acid PEG-80 glyceryl, PEG-60 tonsil oil glyceride, PEG-60 evening primrose glyceride, PEG-200 hydrogenated palm oil fatty acid glyceryl and PEG-90 glyceryl isostearate Can do.

  Preferred ethoxylated oils are coconut oil fatty acid PEG-7 glyceryl, PEG-9 cocoglyceride, PEG-40 hydrogenated castor oil, hydrogenated palm oil fatty acid PEG-200 glyceryl. Ethoxylated glycerol fatty acid esters are used in aqueous cleaning formulations for a variety of purposes. Glycerol fatty acid esters with a low degree of ethoxylation (3 to 12 ethylene oxide units) usually serve as fat additives to improve the skin feel after drying, with a degree of ethoxylation of about 30-50 Glycerol fatty acid esters serve as solubility enhancers for nonpolar substances such as perfume oils. Glycerol fatty acid esters with a high degree of ethoxylation are used as thickeners. The side that all these substances have in common is that they give the skin a special feel when diluted with water and used on the skin.

In combination with the photoprotective agents in photoprotectant skin cosmetic preparations, by likewise present invention the use of keratin-binding effector molecules produced by the methods of the present invention and / or the present invention. These cosmetic and / or dermatological photoprotective compositions are used for cosmetic and / or dermatological photoprotection, for the treatment and care of skin and / or hair, and in cosmetic cosmetics. Used as a product. These include, for example, sun cream, sun lotion, sun milk, sun oil, sambalssum, sangel, lip care and lipstick, concealing cream and stick, moisturizing cream, lotion, emulsion, face, body and hand cream, hair Treatments and rinses, hair setting compositions, styling gels, hair sprays, roll-on deodorants or eye wrinkle creams, tanning agents, sunblocks, post tanning preparations. All preparations contain at least one keratin-binding effector molecule and one specific UV filter substance.

  Sun oil is a mixture of different oils, mainly including one or more light protection filters and perfume oils. Oily ingredients are selected according to various cosmetic properties. Oils that provide a smooth and soft feel to the skin, such as mineral oils (e.g. paraffin oil) and fatty acid triglycerides (e.g. peanut oil, sesame oil, avocado oil, medium chain triglycerides), and the absorption of sun oil into the skin. It is mixed with an oil that improves viscosity, reduces stickiness, and makes the oil film permeable to air and water vapor (sweat). These include branched chain fatty acid esters (eg, isopropyl palmitate) and silicone oils (eg, dimethyl silicone). If oils based on unsaturated fatty acids are used, an antioxidant such as tocopherol is added to prevent them from rancidity. Sun oil, which is an anhydrous formulation, usually does not contain preservatives. Sunmilk and suncream are prepared as oil-in-water (O / W) emulsions and as water-in-oil (W / O) emulsions. Depending on the type of emulsion, the properties of the preparation vary greatly. O / W emulsions spread easily on the skin, most are absorbed quickly and in most cases can be easily washed off with water. W / O emulsions are more difficult to apply and make the skin very smooth and therefore appear to be somewhat more tacky, but are more likely to protect the skin from drying out. W / O emulsions are mostly water resistant. In the case of an O / W emulsion, the emulsion base, the selection of an appropriate photoprotective material, and, where appropriate, the use of an aid (eg, a polymer) determines the degree of water resistance. Liquid and cream-like O / W emulsion bases are similar in composition to other emulsions customary in skin care. Sunmilk should smooth skin dry by the sun, water and wind. They should not be sticky because they feel particularly uncomfortable when hot and in contact with sand. Sunscreen compositions are generally based on a carrier comprising at least one oil phase. However, compositions with only an aqueous base are also possible. Oils, oil-in-water emulsions and water-in-oil emulsions, creams and pastes, lip protection stick compositions or grease-free gels are therefore suitable. Suitable emulsions may also be obtained by phase inversion techniques such as in DE-A-19726121, among others O / W macroemulsions, O / W microemulsions, or O / W / O emulsions with surface-coated titanium dioxide particles in dispersed form. Emulsion).

  Conventional cosmetic auxiliaries that can be considered as additives are, for example, (co) emulsifiers, fats and waxes, stabilizers, thickeners, bioactive ingredients, film formers, fragrances, pigments, pearlescent lusters, storage Materials, pigments, electrolytes (eg, magnesium sulfate) and pH regulators. Stabilizers that can be used are, for example, metal salts of fatty acids such as magnesium stearate, aluminum stearate and / or zinc stearate. Bioactive ingredients are understood to mean, for example, plant extracts, protein hydrolysates and complex vitamins. Conventional film formers include, for example, hydrocolloids (such as chitosan, microcrystalline chitosan or quaternized chitosan), polyvinylpyrrolidone, vinylpyrrolidone-vinyl acetate copolymers, acrylic acid series polymers, quaternary cellulose derivatives and the like. It is a compound of this.

  Suitable light filter active ingredients are substances that absorb UV light in the UV-B and UV-A regions. These are understood to mean organic substances that absorb ultraviolet light and can release the absorbed energy again in a longer wave radiation form, for example heat. The organic material may be oil-soluble or water-soluble. A suitable UV filter is, for example, 2,4,6-triaryl-1,3,5-triazine, in which case the aryl group can in each case carry at least one substituent, The substituent is preferably selected from hydroxy, alkoxy, especially methoxy, alkoxycarbonyl, especially methoxycarbonyl and ethoxycarbonyl. Also suitable are p-aminobenzoic acid esters, cinnamic acid esters, benzophenones, camphor derivatives, and pigments that block ultraviolet radiation such as titanium dioxide, talc and zinc oxide. Particular preference is given to pigments based on titanium dioxide.

Oil-soluble UV-B filters that can be used are, for example, the following substances:
3-benzylidene camphor and its derivatives, such as 3- (4-methylbenzylidene) camphor,
4-aminobenzoic acid derivatives, preferably 2-ethylhexyl 4- (dimethylamino) benzoate, 2-octyl 4- (dimethylamino) benzoate and amyl 4- (dimethylamino) benzoate,
Cinnamic acid esters, preferably 2-ethylhexyl 4-methoxycinnamate, propyl 4-methoxycinnamate, isoamyl 4-methoxycinnamate, isopentyl 4-methoxycinnamate, 2-cyano-3-phenylcinnamic 2-ethylhexyl acid (octocrylene),
Salicylic acid ester, preferably 2-ethylhexyl salicylate, 4-isopropylbenzyl salicylate, homomenthyl salicylate,
Derivatives of benzophenone, preferably 2-hydroxy-4-methoxybenzophenone, 2-hydroxy-4-methoxy-4'-methylbenzophenone, 2,2'-dihydroxy-4-methoxybenzophenone,
Benzalmalonic acid ester, preferably 2-ethylhexyl 4-methoxybenzalmalonic acid,
Triazine derivatives such as 2,4,6-trianilino- (p-carbo-2'-ethyl-1'-hexyloxy) -1,3,5-triazine (octyltriazone) and dioctylbutamidotriazone (Uvasorb (Registered trademark) HEB),
Propane-1,3-dione, such as 1- (4-tert-butylphenyl) -3- (4′-methoxyphenyl) propane-1,3-dione.

Suitable water-soluble substances are
2-phenylbenzimidazole-5-sulfonic acid and its alkali metal, alkaline earth metal, ammonium, alkylammonium, alkanolammonium and glucammonium salts,
Sulfonic acid derivatives of benzophenone, preferably 2-hydroxy-4-methoxybenzophenone-5-sulfonic acid and its salts,
Sulfonic acid derivatives of 3-benzylidene camphor, such as 4- (2-oxo-3-bornylidenemethyl) benzenesulfonic acid and 2-methyl-5- (2-oxo-3-bornylidene) sulfonic acid and its salts is there.

  Particular preference is given to using cinnamic acid esters, preferably 2-ethylhexyl 4-methoxycinnamate, isopentyl 4-methoxycinnamate, 2-ethylhexyl 2-cyano-3-phenylcinnamate (octocrylene).

  In addition, derivatives of benzophenone, in particular the use of 2-hydroxy-4-methoxybenzophenone, 2-hydroxy-4-methoxy-4'-methylbenzophenone, 2,2'-dihydroxy-4-methoxybenzophenone, and propane-1,3 The use of -diones such as 1- (4-tert-butylphenyl) -3- (4'-methoxyphenyl) propane-1,3-dione is preferred.

A suitable typical UV-A filter is
Derivatives of benzoylmethane, such as 1- (4′-tert-butylphenyl) -3- (4′-methoxyphenyl) propane-1,3-dione, 4-tert-butyl-4′-methoxydibenzoylmethane or 1-phenyl-3- (4'-isopropylphenyl) propane-1,3-dione, etc.
An aminohydroxy substituted derivative of benzophenone, for example N, N-diethylaminohydroxybenzoyl n-hexylbenzoate.

  UV-A and UV-B filters can of course also be used in mixtures.

Further suitable UV filter materials are shown in the table below:

  In addition to the above two groups of major photoprotective substances, it is also possible to use an antioxidant type of second photoprotective agent that blocks the photochemical reaction chain caused when UV rays penetrate the skin. Typical examples are superoxide dismutase, catalase, tocopherol (vitamin E) and ascorbic acid (vitamin C).

  A further group is anti-irritants that have anti-inflammatory effects on skin damaged by UV radiation. Such substances are, for example, bisabolol, phytol and phytantriol.

Also according to the invention is the use of keratin-binding effector molecules according to the invention and / or produced by the method of the invention in combination with inorganic pigments that stop UV radiation in skin cosmetic preparations. It is insoluble or sparingly soluble in water, zinc (ZnO), titanium (TiO _2), iron (e.g., Fe ₂ O _3), zirconium (ZrO _2), silicon (SiO _2), manganese (e.g., MnO), aluminum Selected from the group of (Al ₂ O ₃ ), oxides of cerium (eg Ce ₂ O ₃ ), corresponding mixed oxides of metals and mixtures of such oxides, metal oxides and / or other Pigments based on metal compounds are preferred.

  Here, the inorganic pigments can be present in a coated form, ie surface-treated. This surface treatment may be to impart a thin hydrophobic layer to the pigment by methods known per se, for example as described in DE-A-3314742.

  Suitable repellent active ingredients are compounds that are able to keep certain animals, especially insects, away or away from humans. These include, for example, 2-ethyl-1,3-hexanediol, N, N-diethyl-m-toluamide and the like. Suitable hyperemic substances that promote blood flow through the skin are, for example, shrub pine extract, lavender extract, rosemary extract, pine pine extract, horse chestnut extract, birch leaf extract, hayflower extract And essential oils such as ethyl acetate, camphor, menthol, peppermint oil, rosemary extract, eucalyptus oil. Suitable keratolytic and keratin softening materials are, for example, salicylic acid, calcium thioglycolate, thioglycolic acid and its salts, sulfur, and the like. Suitable anti-dandruff active ingredients are, for example, sulfur, sulfur polyethylene glycol sorbitan monooleate, sulfur ricinol polyethoxylate, zinc pyrithione, aluminum pyrithione and the like. Suitable anti-inflammatory agents that suppress skin irritation are, for example, allantoin, bisabolol, dragosantol, chamomile extract, panthenol and the like.

  The use of keratin-binding effector molecules according to the invention and / or produced by the method of the invention in combination with at least one cosmetically or pharmaceutically acceptable polymer is likewise according to the invention.

  Suitable polymers are, for example, cationic polymers having the INCI name Polyquaternium, such as vinylpyrrolidone / N-vinylimidazolium salt copolymers (Luviquat FC, Luviquat HM, Luviquat Care, Luviquat Care), quaternized with diethyl sulfate. N-vinylpyrrolidone / dimethylaminoethyl methacrylate copolymer (Luviquat PQ11), N-vinylcaprolactam / N-vinylpyrrolidone / N-vinylimidazolium salt copolymer (Luviquat E Hold), cationic cellulose derivative (Polyquaternium-4 And -10), acrylamide copolymers (Polyquaternium-7) and chitosan.

  Suitable cationic (quaternized) polymers are also Merquat (a polymer based on dimethyldiallylammonium chloride), Gafquat (a quaternary polymer formed by reacting polyvinylpyrrolidone with a quaternary ammonium compound), Polymer JR (hydroxyethyl cellulose with cationic groups) and plant-based cationic polymers, for example guar polymers such as Jaguar grades from Rhodia.

  Further suitable polymers are also neutral polymers, for example polyvinylpyrrolidone, N-vinylpyrrolidone and copolymers of vinyl acetate and / or vinyl propionate, polysiloxanes, polyvinylcaprolactam and other copolymers with N-vinylpyrrolidone, polyethyleneimine And salts thereof, polyvinylamine and salts thereof, cellulose derivatives, polyaspartates and derivatives. These include, for example, Luviflex Swing (partially hydrolyzed copolymer of polyvinyl acetate and polyethylene glycol, manufactured by BASF Aktiengesellschaft).

  Suitable polymers are also non-ionic water-soluble or water-dispersible polymers or oligomers (polyvinyl caprolactam, eg Luviskol 0 Plus (BASF), or polyvinyl pyrrolidone and its copolymers, especially vinyl esters such as vinyl acetate. And their copolymers, such as Luviskol VA37 (BASF), polyamides such as those based on itaconic acid and aliphatic diamines (for example described in DE-A-4333238).

  Suitable polymers are also amphoteric or zwitterionic polymers such as octylacrylamide / methyl methacrylate / tert-butylaminoethyl methacrylate-hydroxypropyl methacrylate copolymer available under the name Amphomer (from National Starch), and For example, the zwitterionic polymers disclosed in German patent applications DE3929973, DE2150557, DE2817369 and DE3708451. Acrylamidepropyltrimethylammonium chloride / acrylic acid or methacrylic acid copolymers and their alkali metal and ammonium salts are preferred zwitterionic polymers. Further suitable zwitterionic polymers are methacryloyl ethyl betaine / methacrylic acid copolymers marketed under the name Amersette (AMERCHOL), and hydroxyethyl methacrylate, methyl methacrylate, N, N-dimethylamino methacrylate A copolymer of ethyl and acrylic acid (Jordapon (D)).

  Suitable polymers are also nonionic, siloxane-containing, water-soluble or water-dispersible polymers such as polyether siloxanes such as Tegopren (Goldschmidt).

Likewise according to the invention are acetylsalicylic acid, atropine, azulene, hydrocortisone and derivatives thereof (e.g. hydrocortisone-17-valerate), B and D series vitamins, in particular vitamin B ₁ , vitamin B ₁₂ , vitamin D, vitamin A Or its derivatives (such as retinyl palmitate), vitamin E or its derivatives (such as tocopherol acetate), vitamin C and its derivatives (such as ascorbyl glucoside), niacinamide, panthenol, bisabolol, polidocanol, unsaturated Fatty acids (e.g. essential fatty acids (usually called vitamin F), especially γ-linolenic acid, oleic acid, eicosapentaenoic acid, docosahexaenoic acid and its derivatives), chloramphenicol, caffeine, prostaglandin, thymol , Camphor, Extracts from qualene, vegetables and animals or other products (e.g. evening primrose oil, borage oil or carob seed oil, fish oil, cod liver oil or ceramide and ceramide-like compounds, perfume extract, green tea extract, water lily extract, liquorice Extracts, hamamelis), anti-dandruff active ingredients (e.g. selenium disulfide, zinc pyrithione, piroctone olamine, crimbazole, octopirox, polidocanol and combinations thereof), complex active ingredients (e.g. γ-oryzanol complex active ingredients) And / or the present invention in combination with a skin cosmetic active ingredient (s) advantageously selected from the group consisting of calcium salts (calcium pantothenate, calcium chloride, calcium acetate, etc.) Keratin-binding effector molecules produced by the inventive method Is the use of. It is also advantageous to select an active ingredient from the group of fatty substances, for example pure serine oil, Eucerit® and Neocerit®. One or more active ingredients may be used in particular for the treatment and prevention of signs of intrinsic and / or exogenous skin aging, as well as for the treatment and prevention of the adverse effects of ultraviolet radiation on the skin and hair. Is particularly advantageously selected from the group of NO synthesis inhibitors. A preferred NO synthesis inhibitor is nitroarginine. The one or more active ingredients include catechins; bactic acid esters of catechins; aqueous or organic extracts from plants or plant parts having components of catechins or bile acid esters of catechins (e.g., Theaceae Conveniently further selected from the group comprising a family of plants, in particular the leaves of the species Camellia sinensis (green tea) and the like. These typical ingredients (eg polyphenols or catechins, caffeine, vitamins, sugars, minerals, amino acids, lipids) are particularly advantageous. Catechin is a group of compounds to be understood as hydrogenated flavones or anthocyanidins, derivatives of “catechin” (catechol, 3,3 ′, 4 ′, 5,7-flavanpentaol, 2- (3,4-dihydroxy Phenyl) chroman-3,5,7-triol). Epicatechin ((2R, 3R) -3,3 ′, 4 ′, 5,7-flavanpentaol) is a convenient active ingredient for the purposes of the present invention. Also advantageous are plant extracts with the contents of catechins, especially green tea extracts, e.g. camellia plants, especially Chinese species (Camellia sinensis), Assam species (C. assamica) C. taliensis, C. inawadiensis, and extracts from the leaves of these hybrids with, for example, Camellia japonica. Preferred active ingredients are also (-)-catechin, (+)-catechin, (-)-catechin gallate, (-)-gallocatechin gallate, (+)-epicatechin, (-)-epicatechin, (-) Polyphenols and catechins from the group of -epicatechin gallate, (-)-epigallocatechin, (-)-epigallocatechin gallate.

Flavon and its derivatives (often referred to collectively as “flavones”) are convenient active ingredients for the purposes of the present invention. They are characterized by the following basic structure (indicating substitution positions):

Some of the more important flavones that may preferably be used in the preparations according to the invention are listed in Table 8 below:

Flavones usually occur in nature as glycosylated forms.

According to the present invention, the flavonoid has the general formula:

(Wherein Z ₁ to Z ₇ are independently of each other selected from the group of H group, OH group, alkoxy group and hydroxyalkoxy group, and the alkoxy group or hydroxyalkoxy group is not branched or branched, It may have 1 to 18 carbon atoms and Gly is preferably selected from the group of substances) selected from the group of mono- and oligoglycoside groups.

Furthermore, the active ingredient (s) can also be very conveniently selected from the following groups of hydrophilic active ingredients, in particular:
Α-hydroxy acids such as lactic acid or salicylic acid or salts thereof (eg, sodium lactate, lactate Ca, lactate TEA, etc.); urea, allantoin, serine, sorbitol, glycerol, milk protein, panthenol, chitosan.

  The amount of such active ingredient (s) in the preparation according to the invention is preferably from 0.001 to 30% by weight, particularly preferably from 0.05 to 20% by weight, based on the total weight of the preparation, Particularly, it is 1 to 10% by weight. Particular active ingredients and further active ingredients which can be used in the preparations according to the invention are indicated on pages 12-17 of DE 10318526A1, which refers to its full scope at this point.

  Furthermore, the present invention provides for undesirable changes in skin appearance, such as acne or greasy skin, keratosis, rosacea, photosensitivity, inflammatory, erythematous, allergic or autoimmune reactions. It relates to the use of the above preparation to prevent.

  For use, the cosmetic preparations according to the invention are applied to the skin, hair, fingernails or toenails or gums in the manner customary for cosmetic or skin cosmetics.

  The present invention relates to a skin cosmetic comprising a keratin-binding effector molecule, preferably a keratin-binding effector molecule produced by the method of the invention, particularly preferably as described above for its production as a dye, photoprotective agent, vitamin, pro Further provided is a keratin-binding effector molecule using an effector molecule selected from the group consisting of vitamins, carotenoids, antioxidants and peroxide decomposers. Skin cosmetics comprising keratin-binding effector molecules as listed in Table 11 are particularly preferred.

  SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 146, 150, 153, 156, 157, 158, 160, 162, 164, 166, 168 or 170, preferably SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 40, 42, 44, 46, 48, 146, 150, 153, Keratin binding comprising at least one keratin binding polypeptide (ii) according to the sequence shown in 156, 157, 158, 160, 162, 164, 166, 168 or 170, particularly preferably 166 and 168, most preferably 168 Skin cosmetics containing effector molecules are particularly preferred, and the linker molecule (iii) used for the preparation thereof is maleimidocaproic acid. It was. Very particularly preferred is that the linker molecule (iii) used is maleimidocaproic acid, and pantothenic acid, panthenol, an ester of panthenol, an ether of panthenol or a cation-derivatized panthenol is added to the effector molecule (i). The keratin-binding effector molecule described above.

  In a preferred embodiment of the present invention, skin cosmetic or oral care, dental care and denture care compositions, preferably skin and hair treatment compositions are produced according to the invention and / or by the method of the invention. Keratin-binding effector molecules, based on the total weight of the composition, 0.001-1% by weight (wt%), preferably 0.01-0.9% by weight, particularly preferably 0.01-0.8% by weight, or 0.01-0.7% by weight, It is very particularly preferably included at a concentration of 0.01-0.6% by weight, or 0.01-0.5% by weight, most preferably 0.01-0.4% by weight, or 0.01-0.3% by weight. In a further embodiment, the composition comprises 1-10% by weight, preferably 2-8% by weight, of keratin-binding effector molecules according to the invention and / or produced by the method of the invention, based on the total weight of the composition. 3-7% by weight, 4-6% by weight. In a similarly preferred embodiment, the composition comprises 10-20% by weight, preferably 11-19%, of keratin-binding effector molecules according to the invention and / or produced by the method of the invention, based on the total weight of the composition. Contains at concentrations of 12%, 12-18%, 13-17%, 14-16% by weight. In a likewise preferred embodiment, the composition comprises 20-30% by weight, preferably 21-29%, of keratin-binding effector molecules according to the invention and / or produced by the method of the invention, based on the total weight of the composition. Containing in concentrations of wt%, 22-28 wt%, 23-27 wt%, 24-26 wt%.

  The composition according to the invention is preferably a skin protection composition, skin care composition, skin cleansing composition, hair protection composition, hair care composition, hair cleansing composition, hair coloring agent, mouthwash and mouthwash, or decoration Depending on the field of use, these are preferably used in the form of ointments, creams, emulsions, suspensions, lotions, milks, pastes, gels, foams or sprays.

  In addition to keratin-binding effector molecules according to the invention and / or produced by the method of the invention, the skin cosmetics of the invention comprise polymers, pigments, humectants, oils, waxes, enzymes, minerals, vitamins, sunscreens. , Pigments, fragrances, antioxidants, preservatives and / or all of the pharmaceutically active ingredients already listed above.

Furthermore, the following applies to the skin cosmetics of the present invention:
The formulation base of the composition according to the invention preferably comprises cosmetic or skin cosmetic / pharmaceutically acceptable auxiliaries. Pharmaceutically acceptable auxiliaries are auxiliaries known for use in the pharmaceutical field, food technology and related fields, particularly listed in the relevant pharmacopoeia (e.g. DAB Ph.Eur.BP NF). And other auxiliaries whose properties do not interfere with physiological applications.

  Suitable auxiliaries are glidants, wetting agents, emulsifiers and suspending agents, preservatives, antioxidants, anti-irritants, chelating agents, emulsion stabilizers, film formers, gel formers, odor masking agents, Resin, hydrocolloid, solvent, solubility enhancer, neutralizer, penetration enhancer, pigment, quaternary ammonium compound, fat and super fat, ointment, cream or oil-based substance, silicone derivative, stable Agent, sterilant, propellant, desiccant, opacifier, thickener, wax, softener, white oil. Embodiments in this regard are shown, for example, in Fiedler, HP Lexikon der Hilfsstoffe fur Pharmazie, Kosmetik und angrenzende Gebiete [Lexicon of auxiliaries for pharmacy, cosmetics and related fields], 4th edition, Aulendorf: ECV-Editio-Kantor-Verlag, 1996. Is based on such expertise.

  In order to produce a skin cosmetic composition according to the invention, the active ingredient can be mixed or diluted with suitable auxiliaries (excipients). Excipients can be solid, semi-solid or liquid materials that can serve as a vehicle, carrier or medium for the active ingredient. Further auxiliaries are mixed in a manner known to those skilled in the art, if desired. Furthermore, the polymers and dispersions are suitable in formulations, preferably as coating compositions or binders for solid drug forms, or as auxiliaries within them. They can also be used in creams and as tablet coatings and tablet binders.

  According to a further preferred embodiment, the composition according to the invention is a cosmetic, nail care or cosmetic cosmetic preparation for skin and hair care and protection.

  Suitable skin cosmetic compositions are, for example, facial tonics, face masks, deodorants and other cosmetic lotions. Compositions for use in decorative cosmetics include, for example, concealer sticks, stage makeup, mascara and eyeshadows, lipsticks, coal pencils, eyeliners, blushers, powders and eyebrow pencils.

  In addition, keratin-binding effector molecules according to the present invention and / or produced by the method of the present invention can be used for pore packs for cleansing pores, anti-acne compositions, repellents, shaving compositions, post-shave and pre-shave care. Used in cosmetic compositions, after-sun care compositions, hair removal compositions, hair colorants, intimate care compositions, foot care compositions, and baby care.

  Skin care compositions according to the present invention are in particular W / O or O / W skin creams, day creams and night creams, eye creams, face creams, wrinkle removing creams, sunscreen creams, moisturizing creams, bleaching creams, self-tanning creams, Vitamin cream, skin lotion, care lotion and moisturizing lotion.

The dermocosmetic and dermatological compositions according to the present invention, in addition to the keratin-binding effector molecules produced by the method of the present invention and / or the method of the present invention, can be applied to oxidation processes and related aging processes or to the skin and / or hair. As protection against damage, active ingredients that decompose free radicals can also be included. These active ingredients are preferably the substances described in patent applications WO / 0207698 and WO / 03059312, the contents of which are hereby specified, preferably the boron-containing compounds described therein Thereby reducing the peroxide or hydroperoxide, and the corresponding alcohol can be obtained without forming the subsequent state of the free radical. In addition, hindered amines according to general formula 3 can be used for this purpose:

In the formula, the group Z has the following meanings: H; C ₁ ~C ₂₂ alkyl group, preferably methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec- butyl, tert- butyl, pentyl, isopentyl, neopentyl, tert- pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, C ₁ -C ₁₂ alkyl groups such as dodecyl; C ₁ ~C ₂₂ - alkoxyl group, preferably alkoxymethyl, alkoxyethyl, alkoxypropyl, alkoxy isopropyl, alkoxy butyl, alkoxy isobutyl, alkoxy -sec- butyl, Alkoxy-tert-butyl, alkoxypentyl, alkoxyisopentyl, alkoxyneopentyl, alkoxy-tert-pentyl, alkoxyhexyl, alkoxyheptyl, alkoxyoctyl, alkoxynonyl, alkoxydecyl, alkoxyundecyl, al Kishidodeshiru such C ₁ -C ₁₂ of - alkoxyl group; can be phenyl groups are substituted by C ₁ -C ₄ alkyl groups, C ₆ -C, such as phenyl and naphthyl ₁₀ - aryl radical; as above C ₁ C ₆ -C ₁₀ -O- which can be substituted by a -C ₂₂ alkyl group or a C ₁ -C ₂₂ -alkoxy group, preferably by a C ₁ -C ₁₂ alkyl group or a C ₁ -C ₁₂ -alkoxy group An aryl group.

Further, the groups R ^{1 to} R ⁶ have the following meanings independently of each other. _{H, OH, O, C 1} ~C 22 alkyl group, preferably a C ₁ -C ₁₂ alkyl group (methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec- butyl, tert- butyl, pentyl, isopentyl, neopentyl, tert- pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, etc.), C ₁ ~C ₂₂ - alkoxy groups, preferably C ₁ -C ₁₂ - alkoxy group (alkoxymethyl, alkoxyethyl, alkoxypropyl, alkoxy Isopropyl, alkoxybutyl, alkoxyisobutyl, alkoxy sec-butyl, alkoxy-tert-butyl, alkoxypentyl, alkoxyisopentyl, alkoxyneopentyl, alkoxy-tert-pentyl, alkoxyhexyl, alkoxyheptyl, alkoxyoctyl, alkoxynonyl, alkoxydecyl , Alkoxyundecyl, alkoxydodecyl, etc.), C ₆ -C ₁₀ -aryl groups such as phenyl and naphthyl, where the phenyl group can be substituted by a C ₁ -C ₄ alkyl group, C _1- C ₆ -C ₁₀ -O-aryl group which may be substituted by a C ₂₂ alkyl group or a C ₁ -C ₂₂ -alkoxyl group, preferably by a C ₁ -C ₁₂ alkyl group or a C ₁ -C ₁₂ -alkoxyl group.

  The sterically hindered amine 3-dodecyl-N- (2) in an amount of 0.001 to 1 wt% (wt%), preferably 0.01 to 0.1 wt%, 0.1 to 1 wt%, based on the total weight of the composition. , 2,6,6-tetramethyl-4-piperidinyl) succinimide, 3-dodecyl-N- (1,2,2,6,6-pentamethyl-4-piperidinyl) succinimide, 3-octyl-N- (2, 2,6,6-tetramethyl-4-piperidinyl) succinimide, 3-octyl-N- (1,2,2,6,6-pentamethyl-4-piperidinyl) succinimide, 3-octenyl-N- (2,2 , 6,6-tetramethyl-4-piperidinyl) succinimide, 3-octenyl-N- (1,2,2,6,6-pentamethyl-4-piperidinyl) succinimide, and / or use of Uvinul® 5050H Is particularly preferred.

  In addition to the above-described compounds of the invention and suitable carriers, the skin cosmetic preparations can also contain further active ingredients and auxiliaries that are customary in skin cosmetics as described above. These preferably include emulsifiers, preservatives, perfume oils, cosmetic active ingredients such as phytantriol, vitamins A, E and C, retinol, bisabolol, panthenol, photoprotective agents, bleaching agents, coloring agents, coloring agents. , Tanning agents, collagen, protein hydrolysates, stabilizers, pH regulators, dyes, salts, thickeners, gel formers, viscosity modifiers, silicones, wetting agents, fat additives and / or further conventional additives Is mentioned.

  Preferred oil and fat components of skin cosmetics and skin cosmetic compositions include the mineral and synthetic oils described above (e.g., paraffin, silicone oil, etc.), and aliphatic hydrocarbons having more than 8 carbon atoms, animal and vegetable oils (e.g., Sunflower oil, coconut oil, avocado oil, olive oil, lanolin) or wax, fatty acid, fatty acid ester (e.g., triglycerides of C6-C30 fatty acid), wax ester (e.g., jojoba oil, etc.), fatty alcohol, petroleum jelly, hydrogenated Lanolin and acetylated lanolin, and mixtures thereof.

  Skin cosmetics and skin cosmetic preparations are based on silicone compounds in order to obtain specific properties such as texture, diffusion behavior, water resistance and / or improved binding of auxiliaries such as active ingredients and pigments Conditioning materials can also be included.

  Suitable silicone compounds are, for example, polyalkyl siloxanes, polyaryl siloxanes, polyarylalkyl siloxanes, polyether siloxanes or silicone resins.

  Cosmetic or skin cosmetic preparations are prepared by conventional methods known to those skilled in the art.

  Preferably, the cosmetic and skin cosmetic composition is present in the form of an emulsion, in particular a water-in-oil (W / O) or oil-in-water (O / W) emulsion.

  However, other types of formulations such as gels, oils, oleogels, multilayer emulsions (e.g. in the form of W / O / W or O / W / O emulsions), anhydrous ointments or ointment bases etc. may also be selected. It is also possible. Formulations that do not contain emulsifiers, such as aqueous dispersions, hydrogels, or pickering emulsions are also advantageous embodiments.

  The emulsion is produced by a known method. In addition to at least one keratin-binding effector molecule, emulsions are usually conventional ingredients such as fatty alcohols, fatty acid esters, in particular fatty acid triglycerides, fatty acids, lanolin and derivatives thereof, natural or synthetic oils or waxes, and emulsifiers. In the presence of water. The selection of additives specific to the type of emulsion and the preparation of suitable emulsions are described, for example, by Schrader, Grundlagen und Rezepturen der Kosmetika [Fundamentals and formulations of cosmetics], Huthig Buch Verlag, which is expressly incorporated herein by reference. , Heidelberg, 2nd edition, 1989, third part, or Umbach, Kosmetik: Entwicklung, Herstellung und Anwendung kosmetischer Mittel [Cosmetics: development, manufacture and use of cosmetic compositions], 2nd expanded edition, 1995, Georg Thieme Verlag, ISBN 3 13 712602 9, pages 122 ff.

  For example, suitable emulsions in the form of W / O emulsions for skin creams and the like generally comprise an aqueous phase that is emulsified in an oil or fatty phase using a suitable emulsifier system. The polyelectrolyte complex can be used to provide an aqueous phase.

  Preferred fat components that may be present in the fatty phase of the emulsion are hydrocarbon oils such as paraffin oil, pure serine oil, perhydrosqualene and a solution of microcrystalline wax in these oils; sweet tonsil oil, avocado oil, Animal or vegetable oils such as oil, lanolin and its derivatives, castor oil, sesame oil, olive oil, jojoba oil, carite oil, walrus oil; for example, distillation start point is about 250 ° C. under atmospheric pressure such as petrolatum oil, distillation end point A mineral oil having a temperature of 410 ° C .; alkyl myristate, such as isopropyl myristate, butyl myristate or cetyl myristate, hexadecyl stearate, ethyl palmitate or isopropyl palmitate, octanoic acid triglyceride or decanoic acid triglyceride and cetyl ricinoleate Which is a saturated or esters of unsaturated fatty acids.

  The fatty phase may also contain silicone oils that are soluble in other oils, such as dimethylpolysiloxane, methylphenylpolysiloxane and silicone glycol copolymers, fatty acids and fatty alcohols.

  In addition to the above compounds according to the invention, skin care compositions include, for example, carnauba wax, candelilla wax, beeswax, microcrystalline wax, ozokerite wax and oleic acid, myristic acid, linoleic acid, calcium stearate, Mg And waxes such as Al can also be included.

  Furthermore, the emulsion of the present invention may be in the form of an O / W emulsion. Such emulsions usually comprise an oil phase, an emulsifier that stabilizes the oil phase in the aqueous phase, and an aqueous phase that is usually present in a viscous form. Suitable emulsifiers are preferably O / W emulsifiers such as polyglycerol esters, sorbitan esters or partially esterified glycerides.

  According to a further preferred embodiment, the composition according to the invention is a photoprotective composition, a shower gel, a shampoo formulation or a bath preparation, with a photoprotective preparation being particularly preferred.

  Such a formulation comprises at least one keratin-binding effector molecule according to the invention and / or produced by the method of the invention, usually as an anionic surfactant as a basic surfactant, and as a cosurfactant. Amphoteric and / or nonionic surfactants. Further suitable active ingredients and / or auxiliaries are generally selected from lipids, perfume oils, pigments, organic acids, preservatives and antioxidants, and thickeners / gel formers, skin conditioning agents and wetting agents.

  These formulations advantageously comprise 2 to 50% by weight of surfactant, preferably 5 to 40% by weight, particularly preferably 8 to 30% by weight, based on the total weight of the formulation.

  In cleaning, showering and bathing preparations, all anionic, neutral, amphoteric or cationic surfactants commonly used in body cleaning compositions can be used.

  Suitable anionic surfactants include, for example, alkyl sulfates, alkyl ether sulfates, alkyl sulfonates, alkyl aryl sulfonates, alkyl succinates, alkyl sulfosuccinates, N-alkyl sarcosine salts, acyl Taurate, acyl isothionate, alkyl phosphate, alkyl ether phosphate, alkyl ether carboxylate, alpha olefin sulfonate, especially alkali metal and alkaline earth metal salts such as sodium salt, potassium Salts, magnesium salts, calcium salts, and ammonium and triethanolamine salts. The alkyl ether sulfates, alkyl ether phosphates and alkyl ether carboxylates can have 1 to 10 ethylene oxide or propylene oxide units, preferably 1 to 3 ethylene oxide units in the molecule. .

  These include, for example, sodium lauryl sulfate, ammonium lauryl sulfate, sodium lauryl ether sulfate, ammonium lauryl ether sulfate, sodium lauryl sarcosinate, sodium oleyl succinate, ammonium lauryl sulfosuccinate, sodium dodecylbenzenesulfonate, triethanolamine dodecylbenzenesulfonate Is mentioned.

  Suitable amphoteric surfactants are, for example, alkylbetaines, alkylamidopropylbetaines, alkylsulfobetaines, alkyl glycinates, alkyl carboxyglycinates, alkyl amphoacetates or alkyl propionates, alkyl amphodiacetates or alkyl dipropionates. .

  For example, coco dimethyl sulfopropyl betaine, lauryl betaine, cocamidopropyl betaine or sodium cocoamphopropionate can be used.

  Suitable nonionic surfactants are, for example, aliphatic alcohols or alkylphenols having 6 to 20 carbon atoms in the alkyl chain, which may be linear or branched, and ethylene oxide and / or propylene oxide. It is a reaction product. The amount of alkylene oxide is about 6 to 60 mol per mole of alcohol. Furthermore, alkylamine oxides, mono- or dialkyl alkanolamides, fatty acid esters of polyethylene glycol, ethoxylated fatty acid amides, alkyl polyglycosides or sorbitan ether esters are suitable.

  In addition, cleaning, showering and bathing preparations can include conventional cationic surfactants such as quaternary ammonium compounds, such as cetyltrimethylammonium chloride.

  In addition, shower gel / shampoo formulations may contain thickeners such as sodium chloride, PEG-55, propylene glycol oleate, PEG-120 methyl glucose dioleate and others, as well as preservatives, additional active ingredients and auxiliaries. And can contain water.

Hair Treatment Composition According to a further preferred embodiment, the skin cosmetic according to the invention is a hair treatment composition.

  Preferably, the hair treatment composition according to the invention is in the form of a setting foam, hair mousse, hair gel, shampoo, hair spray, hair foam, finish liquid, permanent neutralizer, hair colorant and bleach or hot oil treatment. is there. Depending on the field used, the hair cosmetic preparation can be applied as (aerosol) spray, (aerosol) foam, gel, gel spray, cream, lotion or wax. Hair sprays here include both aerosol sprays and also pump sprays without propellant gas. Hair foams include both aerosol foams and also pump foams without propellant gas. Hair sprays and hair foams preferably contain most or all water-soluble or water-dispersible ingredients. If the compounds used in the hair sprays and hair foams according to the invention are dispersible in water, they can be applied in the form of an aqueous microdispersion with a particle size of usually 1 to 350 nm, preferably 1 to 250 nm. it can. Here, the solids content of these preparations is usually in the range of about 0.5 to 20% by weight. These microdispersions usually do not require emulsifiers or surfactants for their stabilization.

  Further components are to be understood as meaning customary additives in cosmetics, such as propellants, defoamers, surfactant compounds, ie surfactants, emulsifiers, foam formers and solubilizers. The surface active compounds used may be anionic, cationic, amphoteric or neutral. Further conventional ingredients are also eg preservatives, perfume oils, opacifiers, active ingredients, UV filters, care substances (panthenol, collagen, vitamins etc.), protein hydrolysates, α- and β-hydroxycarboxylic acids, Stabilizers, pH modifiers, dyes, viscosity modifiers, gel formers, salts, wetting agents, fat addition agents, complexing agents and further conventional additives may be used.

  Also included herein are all styling and conditioner polymers known in cosmetics that can be used in combination with the keratin-binding effector molecules of the present invention if very specific properties are to be obtained.

  Suitable conventional hair cosmetic polymers are, for example, the above-mentioned cationic, anionic, neutral, nonionic and amphoteric polymers, which are referred to herein.

  In order to establish certain properties, the preparation can also contain conditioning substances based on silicone compounds. Suitable silicone compounds are, for example, polyalkyl siloxanes, polyaryl siloxanes, polyarylalkyl siloxanes, polyether siloxanes, silicone resins or amino-functional silicone compounds such as dimethicone copolyol (CTFA) and amodimethicone (CTFA).

  A propellant is a propellant commonly used in hair sprays or aerosol foams. Propane / butane mixtures, pentane, dimethyl ether, 1,1-difluoroethane (HFC-152a), carbon dioxide, nitrogen or compressed air are preferred.

  Emulsifiers that can be used are all emulsifiers usually used in hair foams. Suitable emulsifiers may be nonionic, cationic or anionic or amphoteric. Examples of nonionic emulsifiers (INCI nomenclature) are laureth such as laureth-4, ceteth such as ceteth-1, polyethylene glycol cetyl ether, ceteareth such as ceteareth-25, polyglycol fatty acid glycerides, hydroxylated lecithin, fatty acids Lactyl ester, alkyl polyglycoside.

  Examples of cationic emulsifiers are cetyldimethyl-2-hydroxyethylammonium dihydrogen phosphate, cetyltrimonium chloride, cetyltrimonium bromide, cocotrimonium methyl sulfate, quaternium-1-x (INCI) It is.

  Anionic emulsifiers are, for example, alkyl sulfates, alkyl ether sulfates, alkyl sulfonates, alkyl aryl sulfonates, alkyl succinates, alkyl sulfosuccinates, N-alkyl sarcosine salts, acyl taurates, Acyl isethionates, alkyl phosphates, alkyl ether phosphates, alkyl ether carboxylates, alpha olefin sulfonates, especially alkali metal salts and alkaline earth metal salts, such as sodium, potassium, magnesium, calcium The salt can be selected from the group of ammonium salts and triethanolamine salts. The alkyl ether sulfates, alkyl ether phosphates and alkyl ether carboxylates can have 1 to 10 ethylene oxide or propylene oxide units, preferably 1 to 3 ethylene oxide units in the molecule. .

  The gel formers that can be used are all gel formers customary in cosmetics. These include slightly cross-linked polyacrylic acids such as Carbomer (INCI), cellulose derivatives such as hydroxypropylcellulose, hydroxyethylcellulose, cationically modified cellulose, polysaccharides such as xanthan gum, caprylic / capric acid Triglycerides, sodium acrylate copolymer, Polyquaternium-32 (and) liquid paraffin (INCI), sodium acrylate copolymer (and) liquid paraffin (and) PPG-1Trideceth-6, acrylamidopropyltrimonium chloride / acrylamide copolymer, steareth-10 allyl Ether, acrylic acid copolymer, Polyquaternium-37 (and) liquid paraffin (and) PPG-1Trideceth-6, Polyquaternium-37 (and) propylene glycol dicaprylate / dicaprylate (and) PPG-1Trideceth-6, Polyquaternium-7, Polyquaternium -44 mentioned That.

  In the shampoo formulation, all anionic, neutral, amphoteric or cationic surfactants commonly used in shampoos can be used.

  Suitable anionic surfactants include, for example, alkyl sulfates, alkyl ether sulfates, alkyl sulfonates, alkyl aryl sulfonates, alkyl succinates, alkyl sulfosuccinates, N-alkyl sarcosine salts, acyl Taurates, acyl isothionates, alkyl phosphates, alkyl ether phosphates, alkyl ether carboxylates, alpha olefin sulfonates, in particular alkali metal salts and alkaline earth metal salts, such as sodium salts, Potassium, magnesium, calcium, and ammonium and triethanolamine salts. The alkyl ether sulfates, alkyl ether phosphates and alkyl ether carboxylates can have 1 to 10 ethylene oxide or propylene oxide units, preferably 1 to 3 ethylene oxide units in the molecule. .

  Suitable are, for example, sodium lauryl sulfate, ammonium lauryl sulfate, sodium lauryl ether sulfate, ammonium lauryl ether sulfate, sodium lauroyl sarcosinate, sodium oleyl succinate, ammonium lauryl sulfosuccinate, sodium dodecylbenzene sulfonate, triethanol dodecylbenzene sulfonate It is an amine.

  Suitable amphoteric surfactants are, for example, alkylbetaines, alkylamidopropylbetaines, alkylsulfobetaines, alkyl glycinates, alkyl carboxyglycinates, alkyl amphoacetates or alkyl propionates, alkyl amphodiacetates or alkyl dipropionates. .

  For example, cocodimethylsulfopropyl betaine, lauryl betaine, cocamidopropyl betaine or sodium cocamphopropionate can be used.

  Suitable nonionic surfactants are, for example, aliphatic alcohols or alkylphenols having 6 to 20 carbon atoms, which may be linear or branched in the alkyl chain, and ethylene oxide and / or propylene oxide. It is a reaction product. The amount of alkylene oxide is about 6 to 60 mol per mole of alcohol. In addition, alkyl amine oxides, mono- or dialkyl alkanolamides, fatty acid esters of polyethylene glycol, alkyl polyglycosides or sorbitan ether esters are suitable.

  In addition, the shampoo formulation can include conventional cationic surfactants such as quaternary ammonium compounds such as cetyltrimethylammonium chloride.

  Conventional conditioning agents can be used in combination with the keratin-binding effector molecules of the present invention to achieve specific effects in shampoo formulations.

  These include, for example, the above cationic polymers of the INCI name Polyquaternium, in particular vinylpyrrolidone / N-vinylimidazolium salt copolymers (Luviquat FC, Luviquat MS, Luviquat Care), N-vinylpyrrolidone quaternized with diethyl sulfate. / Dimethylaminoethyl methacrylate copolymer (Luviquat D PQ11), N-vinylcaprolactam / N-vinylpyrrolidone / N-vinylimidazolium salt copolymer (Luviquat D Hold), cationic cellulose derivatives (Polyquaternium-4 and -10) Acrylamide copolymer (Polyquaternium-7). In addition, protein hydrolysates can be used, and conditioning materials based on silicone compounds such as polyalkylsiloxanes, polyarylsiloxanes, polyarylalkylsiloxanes, polyether siloxanes or silicone resins can also be used. Further suitable silicone compounds are dimethicone copolyol (CTFA) and amino functional silicone compounds such as amodimethicone (CTFA). In addition, cationic guar derivatives such as Guar Hydroxypropyltrimonium Chloride (INCI) can be used.

  In a further embodiment, the hair cosmetic or skin cosmetic preparation serves for the care and protection of the skin or hair and is an emulsion, dispersion, suspension, aqueous surfactant preparation, milk, lotion, cream, balsam Ointments, gels, granules, powders, stick preparations such as lipsticks, foams, aerosols or sprays. Such formulations are very suitable for topical preparations. Suitable emulsions are oil-in-water emulsions and water-in-oil emulsions or microemulsions.

  In general, hair cosmetic or skin cosmetic preparations are used for application to the skin (topical) or hair. Topical preparations are understood here to mean preparations which are suitable for applying the active ingredient to the skin in a finely dispersed state, preferably in a form which can be absorbed through the skin. Suitable for this purpose are, for example, aqueous solutions and hydroalcoholic solutions, sprays, foams, foam aerosols, ointments, aqueous gels, O / W or W / O type emulsions, microemulsions or cosmetic stick preparations. .

  In a preferred embodiment of the cosmetic composition according to the invention, the composition comprises a carrier. Preferred carriers are water, gas, water-based liquids, oils, gels, emulsions or microemulsions, dispersions, or mixtures thereof. Certain carriers exhibit good skin compatibility. Particularly advantageous for topical preparations are aqueous gels, emulsions or microemulsions.

  Emulsifiers that can be used are nonionic surfactants, zwitterionic surfactants, amphoteric surfactants or anionic emulsifiers. The emulsifier may be present in the composition according to the invention in an amount of 0.1 to 10% by weight, preferably 1 to 5% by weight, based on the composition.

The nonionic surfactant used may be, for example, a surfactant from at least one of the following groups:
Linear fatty alcohols having 8 to 22 carbon atoms, fatty acids having 12 to 22 carbon atoms, and alkylphenols having 8 to 15 carbon atoms in the alkyl group, 2 to 30 mol of ethylene oxide and / Or an adduct with 0-5 mol of propylene oxide,
C _12/18 of adducts of ethylene oxide with glycerol 1 to 30 mol - fatty acid monoesters and diesters,
Glycerol monoesters and diesters and sorbitan monoesters and diesters of saturated and unsaturated fatty acids having from 6 to 22 carbon atoms and their ethylene oxide adducts,
Alkyl monoglycosides and oligoglycosides having 8 to 22 carbon atoms in the alkyl group, and ethoxylated analogs thereof,
An adduct of castor oil and / or hydrogenated castor oil and 15 to 60 mol of ethylene oxide,
Polyol esters, especially polyglycerol esters, such as polyglycerol polyricinoleate, polyglycerol 12-hydroxystearate or polyglycerol dimer (as well as mixtures of compounds from two or more of these substance classes Is suitable),
An adduct of castor oil and / or hydrogenated castor oil and 2 to 15 mol of ethylene oxide,
Linear, branched, unsaturated or saturated C _6/22 fatty acids, ricinoleic acid, 12-hydroxystearic acid and glycerol, polyglycerol, pentaerythritol, dipentaerythritol, sugar alcohols (eg sorbitol), alkyl glucosides Partial esters based on (e.g., methyl glucoside, butyl glucoside, lauryl glucoside), and polyglucoside (e.g., cellulose),
Mono-, di- and trialkyl phosphates, and mono-, di- and / or tri-PEG alkyl phosphates and salts thereof,
Wool wax alcohol,
Polysiloxane-polyalkyl polyether copolymers and corresponding derivatives,
Pentaerythritol, fatty acid, mixed esters of citric acid and fatty alcohol, and / or fatty acids having 6 to 22 carbon atoms, methyl glucose, and polyols, preferably glycerol or poly, as disclosed in DE 1165574 Mixed ester of glycerol and polyalkylene glycol.

  In addition, zwitterionic surfactants can be used as emulsifiers. Zwitterionic surfactant is a term used to refer to a surface active compound having at least one quaternary ammonium group and at least one carboxylic acid group or sulfonic acid group in the molecule. . A particularly suitable zwitterionic surfactant is the so-called betaine, N-alkyl-N, N-dimethylammonium glycinate (e.g. cocoalkyldimethylammonium glycinate), N-acylaminopropyl-N, N- Dimethylammonium glycinate (e.g. cocoacylaminopropyldimethylammonium glycinate) and 2-alkyl-3-carboxylmethyl-3-hydroxyethyl imidazoline (in either case 8-18 in alkyl or acyl groups) And cocoacil aminoethyl hydroxyethyl carboxymethyl glycinate. The fatty acid amide derivative known under the CTFA name cocamidopropyl betaine is particularly preferred.

Likewise suitable emulsifiers are amphoteric surfactants. The amphoteric surfactant contains, in addition to the C _8/18 -alkyl group or -acyl group, at least one free amino group and at least one -COOH- group or -SO ₃ H group in the molecule. Is understood to mean a surface-active compound capable of forming Examples of suitable amphoteric surfactants are N-alkylglycine, N-alkylpropionic acid, N-alkylaminobutyric acid, N-alkyliminodipropionic acid, N-hydroxyethyl-N-alkylamidopropylglycine, N-alkyl Taurine, N-alkyl sarcosine, 2-alkylaminopropionic acid and alkylaminoacetic acid (in each case having about 8-18 carbon atoms in the alkyl group).

Particularly preferred amphoteric surfactants are N-cocoalkylaminopropionate, _{cocoacylaminoethylaminopropionate} and C _{12/18 -acyl} sarcosine. In addition to amphoteric emulsifiers, quaternary emulsifiers are also suitable, ester quat type, preferably methyl quaternized difatty acid triethanolamine ester salts being particularly preferred. Furthermore, anionic emulsifiers that can be used are alkyl ether sulfates, monoglyceride sulfates, fatty acid sulfates, sulfosuccinates and / or ether carboxylic acids.

Suitable oil bodies are Gerve alcohols based on fatty alcohols having 6 to 18, preferably 8 to 10 carbon atoms; linear C ₆ -C ₂₂ -fatty acids and linear C ₆ -C ₂₂ - esters of fatty alcohols; branched C ₆ -C ₁₃ - carboxylic acids with linear C ₆ -C ₂₂ - esters of fatty alcohols; linear C ₆ -C ₂₂ - fatty acids with branched alcohols, In particular esters with 2-ethylhexanol; esters of linear and / or branched fatty acids with polyhydric alcohols (e.g. propylene glycol, dimerdiol or trimertriol) and / or Gerve alcohol; C fatty liquid mono- / di-based, triglyceride mixtures _{- C 6 ~C 18;; -} 6 ~C 10 triglycerides based on fatty acid C ₆ -C ₂₂ - and fatty alcohols and / or Guerbet alcohols, aromatic carboxylic acids Especially cheap Esters of Kosan; C ₂ ~C ₁₂ - and dicarboxylic acids, 1 to 22 straight-chain or branched alcohols or 2-10 carbon atoms and 2 to 6 hydroxyl groups, having a carbon atom esters of polyols having; vegetable oils; branched primary alcohols, substituted cyclohexane; linear C ₆ -C ₂₂ fatty alcohols carbonate; Guerbet carbonates; benzoic acid with linear and / or branched C ₆ -C ₂₂ - esters of alcohols (e.g., Finsolv (TM) TN); silicone oils and / or aliphatic or naphthenic hydrocarbons; dialkyl ethers; ring-opening products of epoxidized fatty acid esters with polyols. Oil bodies that can be used are also silicone compounds such as dimethylpolysiloxanes, methylphenylpolysiloxanes, cyclic silicones, and amino-, fatty acid-, alcohol-, polyether-, epoxy-, fluorine-, alkyl- and / or A glycoside-modified silicone compound, which may be in liquid form at room temperature or in resin form. Oil bodies may be present in the composition according to the invention in an amount of 1 to 90% by weight, preferably 5 to 80% by weight and in particular 10 to 50% by weight, based on the composition.

  The list of specific components that can be used with the keratin-binding effector molecules according to the present invention and / or produced by the methods of the present invention should, of course, not be considered exhaustive or limiting. The components can be used individually or in combination with each other.

The present invention further provides a method of applying a skin cosmetic active ingredient to the skin, hair, and / or fingernails or toenails, wherein:
(l) coupling the skin cosmetic active ingredient to a keratin-binding polypeptide;
The keratin-binding effector molecule according to (m) (k) is applied to the skin, hair and / or fingernails or toenails as a component of a skin cosmetic preparation.

Furthermore, the present invention provides a method for increasing the residence time of a skin cosmetic active ingredient on the skin, hair, and / or fingernails or toenails, wherein:
(n) coupling a skin cosmetic active ingredient to a keratin-binding polypeptide;
(o) applying the keratin-binding effector molecule according to (m) as a component of a skin cosmetic preparation to the skin, hair, and / or fingernails or toenails,
(p) the active ingredient is indirectly bound to the skin, hair, and / or fingernails or toenails via the keratin binding domain (in this method, the active ingredient is bound to the keratin binding polypeptide). Residence time is at least 20% or 30%, preferably at least 40%, 50% or 60% compared to free active ingredient (not bound to keratin-binding polypeptide) under otherwise equivalent conditions Particularly preferably at least 75%, 100% or 125%, very particularly preferably at least 150%, 200% or 250%, most preferably 500%, 750% or 1000%).

The present invention further provides compounds of formulas 2, 4 and 5.

In the formula, “n” is an integer of 0-20, preferably 3-15, particularly preferably 3-10, very particularly preferably 3-8, most preferably of all 5. Particularly preferred here are compounds of the formula 2.

  In a further embodiment of the invention, a mixture of specific compounds can be used in the method of the invention. Here, it is also possible to use analogues in which the remaining hydroxy groups are more esterified and / or mixtures thereof.

Array

Experimental Examples The following examples are disclosed to illustrate preferred embodiments of the invention. These examples should not be considered as exhaustive or as limiting the subject of the invention.

The following abbreviations are used in the experimental description:
(2-amino-2-methylpropanol) AMP, (degrees Celsius) ° C, (ethylenediaminetetraacetic acid) EDTA, (hindered amine stabilizer) HAS, (1,1-difluoroethane) HFC152, (international cosmetic ingredient nomenclature) INCI , (Milliliter) ml, (min) min, (oil / water) O / W, (polyethylene glycol) PEG-25, (paraaminobenzoic acid) PABA, (parts per million) ppm, (sufficient amount) qs, ( (Vinyl pyrrolidone) VP, (water / oil) W / O, (active ingredient) AI, (polyvinyl pyrrolidone) PVP, (keratin binding domain) KBD, (keratin binding domain B of human desmoplakin) KBD-B, (human desmo Plakin's keratin-binding domain C) KBD-C, (Keratin-binding domain of human plakophilin) KBD-D.

Example 1: Expression vectors and production strains Various expression vectors were tested for expression of the keratin binding domain (KBD). For this purpose, various promoters (eg IPTG inducible, rhamnose inducible, arabinose inducible, methanol inducible, constitutive promoter, etc.) were used. Similarly, constructs expressing KBD as a fusion protein (eg, as a fusion with thioredoxin, or eGFP, or YaaD [B. subtilis, SWISS-PROT: P37527, PDX1], etc.) were also tested. Here, using various expression systems, both the described KBD-B (keratin binding domain B, SEQ ID NO: 4) and KBD-C (keratin binding domain C, SEQ ID NO: 10), as well as two types of KBD-BC, a combination of domains, was expressed. The mentioned vector constructs are not intended to limit the scope of the claims.

  Representative examples include vector maps of the IPTG inducible vector pQE30-KBD-B (FIG. 1), the methanol inducible vectors pLib15 (FIG. 2) and pLib16 (FIG. 3), and the inducible vector pLib19 (FIG. 4). The procedure for KBD-C can also be similar to the vector construction and expression described.

  For production of KBD, various production hosts such as E. coli strains were used (see Example 2, eg, XL10-Gold [Stratagene], BL21-CodonPlus [Stratagene], etc.). However, other bacterial production hosts such as Bacillus megaterium or Bacillus subtilis were also used. In the case of KBD expression in macrophages, Barg, H., Malten, M. & Jahn, D. (2005). Protein and vitamin production in Bacillus megaterium. In Methods in Biotechnology-Microbial Products and Biotransformations (Barredo, J.-L ., ed, 205-224).

  The fungal production strains used were Pichia pastoris (see Example 3. For example, GS115 and KM71 [both from Invitrogen], etc.), and Aspergillus nidulans (implemented) See Example 4. For example, RMS011 [Stringer, MA, Dean, RA, Sewall, TC, Timberlake, WE (1991) Rodletless, a new Aspergillus developmental mutant induced by direct gene activation. Genes Dev 5: 1161-1171] And SRF200 [Karos, M, Fischer, R (1999) Molecular characterization of HymA, an evolutionarily highly conserved and highly expressed protein of Aspergillus nidulans. Mol Genet Genomics 260: 510-521]. However, other fungal production hosts such as Aspergillus niger (KB0 expression similar to EP0635574A1 and / or WO98 / 46772) may also be used for KBD expression.

Example 2: Expression of KBD in an E. coli strain using, for example, the IPTG-inducible promoter with the expression plasmid pQE30-KBD-B involves various production hosts such as various E. coli strains (eg XL10-Gold [Stratagene , BL21-CodonPlus [Stratagene], etc.), giant fungi, Bacillus subtilis, etc. were used.

  As an illustrative illustration, described herein is the cloning of KBD-B and the expression of KBD-B by E. coli transformed with pQE30-KBD-B.

Cloning of pQE30-KBD-B・ Lambda-MaxiDNA (DNA-Lambda Maxi Kit, manufactured by Qiagen), cDNA bank of human keratinocytes (BD Bioscience, manufactured by Clontech, human keratinocyte cDNA, foreskin, logarithmic phase Prepared from primary culture, vector: λgt11).

PCR was performed using the following oligonucleotides:
Bag43 (5'-GGTCAGTTACGTGCAGCTGAAGG-3 ') (SEQ ID NO: 141), and
Bag44 (5′-GCTGAGGCTGCCGGATCG-3 ′) (SEQ ID NO: 142).

50 μl PCR mix:
10x PCR buffer Pfu Ultra High Fidelity: 5μl
Lambda DNA (744ng / μl) 1μl (1:30 dilution)
dNTP mix (10 mM) 1 μl
Oligo Bag43 (192ng / μl) 0.5μl
Oligo Bag44 (181ng / μl) 0.5μl
Pfu Ultra High Fidelity Polymerase 1μl
41 μl of H ₂ O.

Temperature program:

The obtained PCR product having a size of about 1102 bp was cut out from the agarose gel and purified.
• The purified PCR product was used as a template and then a second round of PCR was performed.
Oligonucleotides used:
Bag53: (5'-CGCGCCTCGAGCCACATACTGGTCTGC-3 ') (SEQ ID NO: 143), and
Bag51 (5′-GCTTAGCTGAGGCTGCCGGATCG-3 ′) (SEQ ID NO: 144).

50 μl PCR mix:
10x PCR buffer TAQ: 5μl
3.5 μl template from above PCR
dNTP mix (10 mM) 1 μl
Oligo Bag53 (345ng / μl) 0.5μl
Oligo Bag51 (157 ng / μl) 0.5 μl
TAQ polymerase 1μl
39 μl of H ₂ O.

Temperature program:

The obtained PCR product having a size of about 1073 bp was cut out from an agarose gel, purified, and cloned into the following vector: pCR2.1-TOPO (manufactured by Invitrogen).
The resulting vector pCR2.1-TOPO + KBD-B (5027 bp) was then transformed, amplified in E. coli, cut with XhoI and EcoRI, and the resulting KBD-B fragment was pBAD / HisA (Invitrogen, similarly cut with XhoI and EcoRI).
The newly formed vector pBAD / HisA + KBD-B (5171 bp) was cut again with SacI and StuI, and the resulting KBD-B fragment was cloned into pQE30 (Qiagen, cut with SacI and SmaI). . The resulting expression vector pQE30-KBD-B (4321 bp, see also FIG. 1) was used for the following KBD-B expression.

  KBD-B (SEQ ID NO: 4) expressed in E. coli with the vector pQE30-KBD-B further contained amino acids MRGSHHHHHHGSACEL at the N-terminus and amino acids GVDLQPSLIS (SEQ ID NO: 166) at the C-terminus.

Expression of KBD-B in E. coli with pQE30-KBD-B • E. coli strain transformed with pQE30-KBD-B (eg XL10-Gold [Stratagene]) was inoculated into a preculture from a plate or glycerol culture . Depending on the scale of the main culture, inoculation with medium (approximately 1: 100) was performed in test tubes or small flasks.
Antibiotics were used according to the strain used (ampicillin 100 μg / ml for pQE30-KBD-B).
Incubation was performed at 250 rpm and 37 ° C.
• The main culture was inoculated with the preculture at about 1: 100. Main culture: LB medium or appropriate minimal medium with corresponding antibiotics. Incubate at 250 rpm and 37 ° C.
-Expression induction by 1 mM IPTG was performed at an optical density (600 nm) exceeding 0.5.
• Cells were centrifuged 4 hours after expression induction.

  The procedure in the fermentor was similar, but expression induction could be performed with much higher OD units, and thus cell and protein yields could be greatly increased.

Example 3: Intracellular and secretory expression of KBD by Pichia pastoris strains (eg, with expression plasmids pLib15 and pLib16) using a methanol-inducible promoter (shaking flask)
Various Pichia pastoris strains such as GS115 and KM71 were used for KBD expression (Pichia Expression Kit, Version M, manufactured by Invitrogen Life Technologies).

  As a representative example, the expression of KBD-B by Pichia pastoris transformed with pLib15 (intracellular expression vector, see FIG. 2) or pLib16 (secreted expression vector, see FIG. 3) is described. To do.

For construction of pLib15, a DNA fragment (SEQ ID NO: 145) encoding KBD-B with a size of 948 bp was converted into oligonucleotides Lib148 (5'-GCTAAGGAATTCACCATGCATCACCATCACCATCACGAGCCACATACTGGTCTGCT-3 '(SEQ ID NO: 147)) and Lib149 (5'- GCTGGAGAATTCTCAGCTAATTAAGCTTGGCTGCA-3 ′ (SEQ ID NO: 148)) and vector pQE30-KBD-B (Example 2, FIG. 1) were used as templates and amplified by PCR. At this time, EcoRI restriction sites were introduced at both ends of the PCR product.
For the construction of pLib16, a DNA fragment (SEQ ID NO: 149) encoding KBD-B with a size of 942 bp was converted into oligonucleotides Lib149 (5'-GCTGGAGAATTCTCAGCTAATTAAGCTTGGCTGCA-3 '(SEQ ID NO: 148)) and Lib150 (5'- GCTAAGGAATTCCATCACCATCACCATCACGAGCCACATACTGGTCTGCT-3 ′ (SEQ ID NO: 151)) and vector pQE30-KBD-B (Example 2, FIG. 1) were used as templates and amplified by PCR. At this time, EcoRI restriction sites were introduced at both ends of the PCR product.

PCR was performed in a 50 μl reaction mixture having the following composition:
1 μl of plasmid-DNA pQE30-KBD-B
1 μl of dNTP-Mix (10 mM each, Eppendorf)
5 μl of 10 × PCR buffer + MgCl ₂ (Roche)
1 μl of Lib148 or Lib150 5 'primer (equivalent to 50 pmol)
1 μl of Lib149 3 'primer (equivalent to 50 pmol)
5U Pwo polymerase (Roche).

PCR reactions were performed under the following cycle conditions:
Step 1: 5 minutes, 95 ° C (denaturation)
Step 2: 45 seconds, 95 ° C
Step 3: 45 seconds, 50 ° C (annealing)
Step 4: 2 minutes, 72 ° C (extension)
30 cycles of steps 2-4
Step 5: 10 minutes, 72 ° C (post-extension)
Step 6: 4 ° C (stop).

A PCR product (SEQ ID NO: 145) amplified by oligonucleotides Lib148 / Lib149 was digested with EcoRI and ligated into a vector pPIC3.5 (Pichia Expression Kit, Version M, manufactured by Invitrogen) cut with EcoRI. The vector pLib15 (FIG. 2) obtained from the ligation was checked for correct KBD-B amplification by sequencing.
A PCR product (SEQ ID NO: 149) amplified by oligonucleotide Lib149 / Lib150 was digested with EcoRI and ligated into a vector pPIC9 (Pichia Expression Kit, Version M, manufactured by Invitrogen) cleaved with EcoRI. The vector pLib16 obtained from ligation (FIG. 3) was sequenced to check for correct KBD-B amplification.
• Pichia pastoris strain electrocompetent cells and spheroplasts were transformed with circular vector and StuI linear vectors pLib15 and pLib16 according to manufacturer's instructions (Pichia Expression Kit, Version M, manufactured by Invitrogen).
Transformants were analyzed by PCR using chromosomal DNA and Southern blot.

• For preculture, Pichia pastoris transformants expressing KBD-B were inoculated from plates or glycerol cultures. Depending on the scale of the main culture, MGY, BMG or BMGY medium (Pichia Expression Kit, Version M, manufactured by Invitrogen) (about 1: 100) in a test tube or small flask was inoculated.
The culture was incubated at 250-300 rpm, 30 ° C. until OD ₆₀₀ = 2-6.
-Cells were collected at 1500-3000 xg for 5 minutes at room temperature.
For the main culture, the recovered cell pellet was added at a concentration of OD ₆₀₀ = 1 in methanol-containing mM, BMM or BMMY medium (Pichia Expression Kit, Version M, manufactured by Invitrogen) to induce expression.

The main culture was incubated for 1 to 96 hours at 250 to 300 rpm and 30 ° C.
Expression induction was maintained every 24 hours by adding 100% methanol at a final methanol concentration of 0.5%.
In the case of intracellular expression, the cells were collected and destroyed using Menton-Gaulin after the main culture was completed.
In the case of secretory expression, the culture supernatant was collected and KBD-B was directly purified therefrom.

KBD-B (SEQ ID NO: 145) (pLib15) expressed intracellularly in Pichia pastoris further contains the amino acid MHHHHHH at the N-terminus and the amino acid GVDLQPSLIS at the C-terminus in addition to the polypeptide sequence (SEQ ID NO: 4) It was.
In KBD-B (SEQ ID NO: 149) (pLib16) secreted and expressed in Pichia pastoris, in addition to the polypeptide sequence (SEQ ID NO: 4), the amino acid MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASHAKEVVSLEH Further included.
KBD-B (SEQ ID NO: 149) (pLib16) processed and secreted by Pichia pastoris further includes the amino acid YVEFHHHHHH at the N-terminus and the amino acid GVDLQPSLIS at the C-terminus in addition to the polypeptide sequence (SEQ ID NO: 4) It was supposed to be.

Example 4: Expression of KBD by an Aspergillus nidulans strain (shaking flask) using an inducible alcA promoter (eg via the expression plasmid pLib19)
For expression, Aspergillus nidulans wild type strains such as RMS011 or SRF200 were used. Hereinafter, as an illustrative explanation, the expression of KBD-B by Aspergillus nidulans transformed with pLib19 is described (FIG. 4).

For the construction of pLib19, a DNA fragment (SEQ ID NO: 152) encoding KBD-B with a size of 922 bp was converted into oligonucleotides Lib151 (5'-CACCATGCATCACCATCACCATCACGAGCCACATACTGGTCTGCT-3 '(SEQ ID NO: 154)) and Lib152 (5'- GCTAATTAAGCTTGGCTGCA-3 ′ (SEQ ID NO: 155)) and vector pQE30-KBD-B (Example 2, FIG. 1) were used as templates to be amplified by PCR (adapted to the Tm values of primers Lib151 and Lib152, 53 Using the above PCR conditions, using the annealing temperature of the PCR program in ° C). The PCR product was ligated into the vector pENTR / D (pENTR ™ Directional TOPO ™ Cloning Kit, Version E, Invitrogen). Correct KBD-B amplification was checked by sequencing.
-Using the `` Gateway (registered trademark) LR clonase (TM) enzyme mix '' (manufactured by Invitrogen), the DNA fragment encoding KBD-B was recombined into the vector pMT-OvE (Toews MW, Warmbold J, Konzack S, Rischitor P, Veith D, Vienken K, Vinuesa C, Wei H, Fischer R; Establishment of mRFP1 as a fluorescent marker in Aspergillus nidulans and construction of expression vectors for high-throughput protein tagging using recombination in vitro ( GATEWAY). (2004) Curr Genet 45: 383-389). This generated the vector pLib19 (Figure 4).

Protoplast of Aspergillus nidulans wild type strain was transformed with circular vector pLib19 (Yelton MM, Hamer JE, Timberlake WE; Transformation of Aspergillus nidulans by using a trpC plasmid., (1984) Proc Natl Acad Sci USA 81: 1479-1474). Transformants were analyzed by PCR using chromosomal DNA and Southern blot.
For pre-culture of Aspergillus nidulans transformants expressing KBD-B, 100 ml of minimal medium (0.6% NaNO ₃ , 0.152% KH ₂ PO ₄ , 0.052% KCl [pH 6. 5], 0.8% glucose, 0.05% MgSO ₄ , 1 ml trace element solution [1 g / l FeSO ₄ × 7H ₂ O, 8.8 g / l ZnSO ₄ × 7H ₂ O, 0.4 g / l CuSO ₄ × 5H ₂ O, 0.15 g / l MnSO ₄ × 4H ₂ O, 0.1 g / l Na ₂ B ₄ O ₇ × 10H ₂ O, 0.05 g / l (NH ₄ ) ₆ Mo ₇ O ₂₄ × 4H ₂ O], + Strain-specific additive) or 100 ml complete medium (2% malt extract, 0.1% peptone, 2% glucose, + strain-specific additive) inoculated with 10 ⁶ to 10 ⁷ spores, 200-250 rpm, Incubated for 16-24 hours at 37 ° C.
• After pre-culture, fungal mycelium was collected by filtration, washed with distilled water and transferred to a flask with 100-500 ml of fresh minimal medium. In this main culture medium, 0.1% fructose was used instead of glucose as a C source. To induce KBD expression, ethanol (1% final concentration) or glycerol (50 mM) or sodium acetate (50 mM) or ethylamine or threonine was further added to the medium. Additives mentioned to induce expression do not limit the scope of the claims. The culture was further incubated at 200-250 rpm and 37 ° C. for 5-48 hours.
-After completion of the culture, the mycelium of the fungus was collected at 1500 to 3000 xg for 5 minutes at room temperature and destroyed using Menton-Gaulin.

In addition to the polypeptide sequence (SEQ ID NO: 4), KBD-B (SEQ ID NO: 152) (pLib19) expressed in Aspergillus nidulans contains amino acids MHHHHHH at the N-terminus and amino acids GVDLQPSLISKGGRADPAFLYKVVMIRLLTKPERKLLEGGPGTQLLFPLVRVNCALGVRK It was.

Example 5: Cell disruption and intracellular inclusion body purification (pQE30-KBD-B)
Soluble expressed KBD could be used directly after cell disruption (eg, by Menton-Gaulin) or purified by chromatography (see Example 6). KBD expressed insoluble (eg, in inclusion bodies) was purified as follows.
-The fermenter contents were centrifuged and the pellet was suspended in 20 mM phosphate buffer (pH = 7.5) and disrupted using Menton-Gaulin.
The disrupted cells were centrifuged again (15000 g) and the pellet was treated with 20 mM phosphate, 500 mM NaCl and 8 M urea and agitated (inclusion body lysis).
-The pH of the supernatant was adjusted to 7.5.
• The centrifugation was then performed again and the supernatant was added to a Ni chelate sepharose column and purified as described in Example 6.

Example 6: Purification of keratin binding domain B with Ni chelate Sepharose KBD could be purified by chromatography on a Ni column via a bound His tag.
Column material: Ni-Sepharose High Performance
Amersham Biosciences, part number 17-5268-02.

The material was packed in a column (eg, 2.6 cm diameter, 10 cm height) and equilibrated with buffer A + 4% buffer B (corresponding to 20 mM imidazole).
Protein extracts (see, eg, cell disruption and intracellular inclusion body purification) were added to the column (pH 7.5) (flow rate, approximately 5 ml / min) using Superloop (AKTA system).
After the addition, washing was performed with buffer A + 20 mM imidazole.
Elution was performed with buffer B (500 mM imidazole in buffer A).
The eluate was collected as fractions using a fraction collector.
The eluate was then desalted (convenient for the sample to be concentrated). For this purpose, the eluate was desalted with, for example, a Sephadex G25 medium size column (manufactured by Amersham). Subsequently, for example, an Amicon chamber (stirred ultrafiltration cell, manufactured by Millipore) was possible for concentration.

Buffer A: 20 mM sodium dihydrogen phosphate
500 mM NaCl (If desired, a buffer with a low concentration of NaCl can be used.)
8M urea (If the already active expressed "active" KBD is separated by chromatography, it is not necessary to use urea. In the absence of urea, subsequent protein regeneration is not necessary.)
pH = 7.50
Buffer B: 20 mM sodium dihydrogen phosphate
500 mM NaCl (If desired, a buffer with a low concentration of NaCl may be used.)
8M urea
500 mM imidazole
pH = 7.50.

Example 7: Regeneration of keratin binding domain B Insoluble expressed keratin binding domains (eg from inclusion bodies) can be regenerated and thus activated as described below.

Method 1: discontinuous dialysis
Cellytic IB (Sigma, product number C5236) 6.5 ml and 5 mM DTT were added to 6.5 ml KBD-B inclusion bodies in 8 M urea (Ni chelate eluent, HiTrap). Next, the solution to be regenerated was poured into a dialysis tube (Spectrum: Spectra Por MWCO: 12-14 kD).
Dialysis was performed against 1 L of 6M urea solution for about 12 hours at 4 ° C. with careful stirring.
500 ml of 25 mM Tris / HCl (pH = 7.50) was added, and dialysis was similarly performed at 4 ° C. for 9 hours. An additional 250 ml of Tris buffer (see above) was then added and dialyzed for an additional 12 hours.

Subsequently, 500 ml of 25 mM Tris / HCl (pH = 7.50) was added again, and dialysis was similarly performed at 4 ° C. for 9 hours. An additional 250 ml of Tris buffer (see above) was then added and dialyzed for an additional 12 hours.
Subsequently, 500 ml of 25 mM Tris / HCl (pH = 7.50) was added again, and dialysis was similarly performed at 4 ° C. for 9 hours. The dialysis tube containing the dialysate was then added into 2 L of 25 mM Tris + 150 mM NaCl (pH = 7.50). Then, dialysis was performed again at 4 ° C. for 12 hours.
The contents of the dialysis tube were then removed.

Method 2: Continuous dialysis
20 ml of KBD-B inclusion bodies in 8M urea (Ni chelate eluate, HiTrap) was treated with 10 ml of Cellytic IB (Sigma, product number C5236) and 5 mM DTT. The solution was then poured into a dialysis chamber (Slide-A-Lyzer Dialyses Cassette PIERCE, MWCO: 10 kD, product number 66830).
Next, dialysis was performed for 1 hour at 4 ° C. against 1 L of 6 M urea solution.
Subsequently, 2 L of the following buffer was continuously fed over 48 hours while metering with a peristaltic pump (25 mM Tris / HCl, pH = 7.5).
The dialysis tube containing the dialysate was then added to 2 L of the final buffer (25 mM Tris + 150 mM NaCl, pH = 7.50) and dialyzed at 4 ° C. for about 12 hours.
The contents of the dialysis tube were then removed.

Example 8: Binding to skin 1 (qualitative)
A visual qualitative test was developed to see if KBD binds to the skin.

Solution used:
Blocking solution: Western blocking reagent 1921673 Roche (10x solution) diluted with TBS
TBS: 20 mM Tris, 150 mM NaCl (pH 7.5)
TTBS: TBS + 0.05% Tween20.

The first step is to transfer the outer stratum corneum of the skin to a stable support. For this purpose, a transparent adhesive tape is applied firmly to the depigmented human skin and peeled off. The test can be performed directly on the transparent adhesive strip or the adhered stratum corneum can be re-adhered to the glass slide and transferred. Binding was demonstrated as follows.
• Transfer to a Falcon container for incubation with various reagents.
• If appropriate, add ethanol for degreasing, remove the ethanol and dry the slides.
Incubate with blocking buffer at room temperature for 1 hour.
・ Wash twice with TTBS for 5 minutes.
-Wash once with TBS for 5 minutes.
Incubate with KBD to be tested (coupled to tags (eg, His ₆ , HA, etc.)) or control protein in TBS / 0.05% Tween 20 for 2-4 hours at room temperature.
Remove the supernatant.
・ Wash 3 times with TBS.
Incubate with monoclonal anti-polyhistidine (or specific KBD rabbit) antibody diluted 1: 2000 in TBS + 0.01% blocking for 1 hour at room temperature.
・ Wash with TTBS twice for 5 minutes.
・ Wash once with TBS for 5 minutes.
Incubate for 1 hour at room temperature with anti-mouse IgG alkaline phosphatase conjugate diluted 1: 5000 in TBS + 0.01% blocking.
・ Wash with TTBS twice for 5 minutes.
・ Wash once with TBS for 5 minutes.
Add phosphatase substrate (NBT-BCIP; Boehringer MA1 tablet / 40 ml water, 2.5 min, stop with water).
• Optically detect colored precipitates with the naked eye or using a microscope. A blue precipitate indicates that KBD has bound to the skin.

Example 9: Binding to skin 2 (quantitative)
A quantitative test has been developed that can compare the hair / skin binding strength of KBD with non-specific proteins.

A 5mm punch was used to puncture a section from a thawed dry piece (human or pig) without hair (or, for surface testing, place a section of skin in a falcon lid). The skin sample is then made 2-3 mm thick and any tissue present is removed. The skin sample is then transferred to an Eppendorf container (low protein binding) to demonstrate binding (see also Figure 6. Alternatively, use the L'Oreal Episkin system [reconstructed human skin]. be able to).
-Wash twice with PBS / 0.05% Tween20.
Add 1 ml of 1% BSA in PBS and incubate for 1 hour at room temperature with gentle rotation (900 rpm).
Remove the supernatant.
Add 100 μg KBD in PBS to 0.05% Tween20 and incubate for 2 hours at room temperature with gentle rotation (900 rpm).
Remove the supernatant.
-Wash 3 times with PBS / 0.05% Tween20.
1 ml monoclonal mouse anti-tag (His6 or HA or specific KBD) antibody (with peroxidase conjugate) (1: 2000 in PBS containing 0.05% Tween20) with gentle rotation (900 rpm) [monoclonal anti Incubate with polyhistidine peroxidase conjugate, produced in mice, lyophilized powder, Sigma] for 2-4 hours at room temperature.
-Wash 3 times with PBS / 0.05% Tween20.
Add peroxidase substrate (1 ml / eppendorf container; see composition below).
-The reaction is allowed to proceed until it turns blue (about 90 seconds).
Stop the reaction with 100 μl of 2M H ₂ SO ₄ .
-Absorption at 405 nm was measured.

Peroxidase substrate (prepared immediately before):
0.1 ml of TMB solution (42 mM TMB in DMSO)
+ Substrate buffer 10ml (0.1M sodium acetate pH4.9)
+14.7 μl H ₂ O ₂ (3% concentration).

Example 10: Binding to hair (quantitative)
A quantitative assay was developed so that the binding strength of KBD to hair compared to other proteins could be shown (see also FIG. 6). In this test, the hair was first incubated with KBD to wash away excess KBD. The antibody-peroxidase conjugate was then coupled via the His tag of KBD. Unbound antibody-peroxidase conjugate was washed off again. Bound antibody-peroxidase conjugate [monoclonal anti-polyhistidine peroxidase conjugate, produced in mice, lyophilized powder, Sigma] can convert colorless substrate (TMB) into a colored product. Can be measured by photometric measurement at 405 nm. Absorption intensity indicates the amount of KBD or comparative protein bound. The selected comparative protein is, for example, YaaD derived from Bacillus subtilis, and is necessary for this test, so that it also has a His tag for detection. Instead of the His tag, other specific antibodies conjugated with peroxidase can also be used.

Hair (human) 5 mg is cut to 5 mm length and transferred to an Eppendorf container (low protein binding) to demonstrate binding.
・ Add 1 ml of ethanol for degreasing.
· Centrifuged to remove ethanol, wash the hair with H ₂ O.
Add 1 ml 1% BSA in PBS and incubate for 1 hour at room temperature with gentle swirling.
• Centrifuge and remove supernatant.
Keratin binding domain tested in PBS / 0.05% Tween20 in 1 ml (tag (e.g., His _6, the HA, etc.) are coupled) or control protein was added, with gentle swirling motion, at 4 ° C. Incubate for 16 hours (or at least 2 hours at room temperature).
• Centrifuge and remove supernatant.
-Wash 3 times with PBS / 0.05% Tween20.
1 ml monoclonal mouse anti-tag (His6 or HA) antibody (with peroxidase conjugate) (in PBS / 0.05% Tween20, 1: 2000) [monoclonal anti-polyhistidine peroxidase conjugate, in mouse with gentle swirl Incubate 2-4 hours at room temperature with production, lyophilized powder, Sigma.
-Wash 3 times with PBS / 0.05% Tween20.
Add peroxidase substrate (1 ml / eppendorf container).
・ Proceed with the reaction until it turns blue (about 2 minutes).
Stop the reaction with 100 μl of 2M H ₂ SO ₄ .
・ Measure the absorption at 405 nm.

Peroxidase substrate (prepared immediately before)
0.1 ml of TMB solution (42 mM TMB in DMSO)
+10 ml substrate buffer (0.1 M sodium acetate, pH 4.9)
+14.7 μl H ₂ O ₂ (3% concentration)
BSA = bovine serum albumin
PBS = phosphate buffered saline
Tween 20 = polyoxyethylene sorbitan monolaurate, n is about 20
TMB = 3,5,3 ′, 5′-tetramethylbenzidine.

In a hair binding test exemplarily performed for KBD-B, the binding property of KBD-B (SEQ ID NO: 166) to hair is very superior compared to significantly insufficient binding of the comparative protein YaaD. It was shown that.

Example 11: Expression of KBD-D (SEQ ID NO: 167) by E. coli strain using expression plasmid pRee024 with IPTG inducible promoter (Figure 8)
E. coli strain XL10 Gold [Stratagene] was used for expression.

  Here, as a representative example, cloning of KBD-D (SEQ ID NO: 167) and subsequent expression of KBD-D protein (SEQ ID NO: 168) in E. coli transformed with pRee024 are described (FIG. 8).

Cloning of pRee024Lambda -MaxiDNA (DNA-Lambda Maxi Kit, Qiagen), cDNA bank of human keratinocytes (BD Bioscience, Clontech, cDNA of human keratinocytes, foreskin, logarithmic primary culture, vector : λgt11).

  PCR for amplification of the KBD-D gene was performed in two steps. First, the 5 ′ end and 3 ′ end were amplified separately. These fragments were a matrix for amplification of the entire KBD-D gene.

PCR for amplification of the 5 ′ end was performed as follows.
The primers had the following sequences:
HRe6: 5'-ATGAACCACTCGCCGCTCAAGACCGCCTTG-3 '(SEQ ID NO: 171)
HRe9: 5′-CGTTCCCGGTTCTCCTCAGGAGGCTGACTG-3 ′ (SEQ ID NO: 172).

100 μl PCR mix:
10x PCR buffer Pfu Ultra High Fidelity: 10μl
Lambda DNA (744ng / μl) 1μl (1:10 dilution)
dNTP mix (10 mM) 10 μl
HRe6 (196ng / μl) 1μl
HRe9 (201ng / μl) 1μl
Pfu Ultra High Fidelity Polymerase 1μl
76 μl of double-distilled H ₂ O.

Temperature program:

A fragment of about 1 kb size was detected in an agarose gel. The reaction was purified and used below as a 5 ′ end template for amplification of the KBD-D gene.

PCR for amplification of the 3 ′ end was performed as follows.
The primers had the following sequences:
HRe7: 5'-TTAGAATCGGGAGGTGAAGTTCCTGAGGCT-3 '(SEQ ID NO: 173)
HRe8: 5′-CACCACCAACAAGCTGGAGACCCGGAG-3 ′ (SEQ ID NO: 174).

100 μl PCR mix:
10x PCR buffer Pfu Ultra High Fidelity: 10μl
Lambda DNA (744ng / μl) 1μl (1:10 dilution)
dNTP mix (10 mM) 10 μl
HRe7 (201ng / μl) 1μl
HRe8 (209ng / μl) 1μl
Pfu Ultra High Fidelity Polymerase 1μl
76 μl of double-distilled H ₂ O.

Temperature program:

-A fragment with a size of about 1.2 kb was detected in an agarose gel. The reaction was purified and used below as a 3 ′ end template for amplification of the KBD-D gene.

• For amplification of KBD-D gene, 5 'and 3' end templates were used as matrix. PCR was performed as follows.
100 μl PCR mix:
10x PCR buffer Pfu Ultra High Fidelity: 10μl
dNTP-mix (10 mM) 10 μl
Double-distilled H ₂ O 75 μl
5 'end template 1μl
3 'end template 1μl
Pfu Ultra High Fidelity Polymerase 1μl
76 μl of H ₂ O.

Temperature program:

After 10 cycles, 1 μl of primers HRe6 (196 μg / ml) and HRe7 (206 μg / ml) and 1 μl of Pfu Ultra High Fidelity polymerase were added and the following temperature program with reaction was performed.
Temperature program:

Then 1 μl Taq polymerase was added and the mixture was incubated at 72 ° C. for 10 minutes.
The obtained PCR product having a size of about 2150 bp was cut out from an agarose gel, purified, and cloned into the following vector pCR2.1-TOPO (manufactured by Invitrogen).
The resulting vector pRee019 (6112bp) was then transformed and amplified in E. coli and the KBD-D gene was examined by sequencing.

The KBD-D gene was then cloned into an expression vector. Therefore, PCR was further performed using the vector pRee019 as a template.
Oligonucleotides used:
HRe26: 5'-CTCGGTACCAACCACTCGCCGCTCAAGACCGCCTTGGCG-3 '(SEQ ID NO: 175)
HRe27: 5′-ATTAAGCTTTTAGAATCGGGAGGTGAAGTTCCTGAGGCT-3 ′ (SEQ ID NO: 176).

100 μl PCR mix:
10x PCR buffer Pfu Ultra High Fidelity: 10μl
pRee019 (25ng / μl) 1μl
dNTP mix (10 mM) 10 μl
HRe26 (287ng / μl) 1μl
HRe27 (354ng / μl) 1μl
Pfu Ultra High Fidelity Polymerase 1μl
76 μl of double-distilled H ₂ O.

Temperature program:

-A fragment with a size of about 2.2 kb was detected in an agarose gel. The reaction was purified and then cut with restriction endonucleases KpnI and HindIII and the resulting fragment was cloned into an expression vector. This resulted in the vector pRee024, which was then used for KBD-D expression.

Expression of KBD-D (SEQ ID NO: 167) by pRee024 in E. coli • An E. coli strain transformed with pRee024 (eg, TG10) was inoculated into a preculture from a plate or glycerol culture. Depending on the scale of the main culture, inoculation with LB medium (approximately 1: 100) was performed in test tubes or small flasks.
• Antibiotics were used according to the strain used (ampicillin 100 μg / ml for E. coli TG10 transformed with pRee024).
Incubated at 250 rpm and 37 ° C.
• The main culture was inoculated with the preculture at about 1: 100. Main culture: LB medium or the appropriate minimal medium with the corresponding antibiotic. Incubation was performed at 250 rpm and 37 ° C.
_-When OD _578nm exceeded 1, expression was induced using 1 mM IPTG. The incubation temperature was then lowered to room temperature (about 20 ° C.). Cells were centrifuged 2 hours after expression induction (see FIG. 9).

Example 12: Cell disruption and intracellular inclusion body purification (pRee024)
KBD-D (SEQ ID NO: 168) expressed insoluble (eg, in inclusion bodies) was purified as follows.
The cell pellet from Example 2 was resuspended in 20 mM phosphate buffer containing 100 mM NaCl (pH = 7.5) and disrupted by sonication.
The disrupted cells were centrifuged again (4 ° C., 12000 g, 20 minutes). The supernatant was discarded. The precipitate was redissolved in buffer A (10 mM NaH ₂ PO ₄ , 2 mM KH ₂ PO ₄ , 100 mM NaCl, 8 M urea, 5 mM DTT). The mixture was then centrifuged again and the supernatant was added to Ni chelate sepharose. After the addition, washing was performed with buffer A 20 mM imidazole. Elution from the column was performed with buffer B (10 mM NaH ₂ PO ₄ , 2 mM KH ₂ PO ₄ , 100 mM NaCl, 8 M urea, 5 mM DTT, 500 mM imidazole). The eluate was collected as a fraction and analyzed by SDS-PAGE. The fraction containing purified KBD-D was regenerated as described in Example 13.

Example 13: Regeneration of keratin binding domain D (SEQ ID NO: 168)
Insoluble expressed keratin binding domain D (eg, from inclusion bodies) could be regenerated by dialysis and thus activated. The procedure was as follows.
The fraction from Example 12 containing purified KBD-D was poured into a dialysis tube (MWCO 12-14 KD).
Next, dialysis was performed for 1 hour against 1 L of 8M urea solution.
Next, 2 L of deionized water was continuously supplied over 12 hours while being metered by a peristaltic pump.
The contents of the dialysis tube were then removed. KBD-D thus activated was used in the activity test described below.

Example 14: Qualitative binding to skin
A visual qualitative test was used to test whether KBD-D (SEQ ID NO: 168) bound to the skin.

Solution used:
Blocking solution: Western blocking reagent 1921673 Roche (10x solution) diluted with TBS
TBS: 20 mM Tris, 150 mM NaCl, pH 7.5
TTBS: TBS + 0.05% Tween20.

The first step is to transfer the outer stratum corneum of the skin to a stable support. For this purpose, a transparent adhesive tape is applied firmly to the depigmented human skin and peeled off. The test can be performed directly on the transparent adhesive strip or the adhered stratum corneum can be re-adhered to the glass slide and transferred. Binding was demonstrated as follows.
• Transfer to a Falcon container for incubation with various reagents.
• If appropriate, add ethanol for degreasing, remove the ethanol and dry the slides.
Incubate with blocking buffer at room temperature for 1 hour.
・ Wash twice with TTBS for 5 minutes.
-Wash once with TBS for 5 minutes.
Incubate with KBD to be tested (coupled to tags (eg, His ₆ , HA, etc.) in TBS / 0.05% Tween 20 for 2-4 hours at room temperature.
Remove the supernatant.
・ Wash 3 times with TBS.
Monoclonal mouse anti-tag (His6 or HA) antibody (with peroxidase conjugate) (TBS + 0.01% in blocking, 1: 2000) [monoclonal anti-polyhistidine peroxidase conjugate, produced in mice, lyophilized powder, Sigma Incubate for 1 hour at room temperature.
・ Wash twice with TTBS for 5 minutes.
-Wash once with TBS for 5 minutes.
Add phosphatase substrate (NBT-BCIP; Boehringer MA1 tablet / 40 ml water, 2.5 min; stop with water).
• Optically detect colored precipitates with the naked eye or using a microscope.

  A blue precipitate, a reaction of anti-polyhistidine-AP conjugate interacting with KBD-D, was seen on the transparent adhesive tape treated with KBD-D. As a negative control, a transparent adhesive tape was treated with buffer only. Clear blue coloration was not seen here. These results indicate that KBD-D bound to skin keratin on transparent adhesive tape.

Example 15: Quantitative binding to skin and hair
To investigate the binding strength of KBD-D (SEQ ID NO: 168) to skin and hair compared to KBD-B (SEQ ID NO: 166), a quantitative test was performed. In this test, the hair was first incubated with KBD-B or KBD-D to wash away excess KBD-B or -D. The antibody-peroxidase conjugate was then coupled via the KBD-B or -D His / tag. Unbound antibody-peroxidase conjugate was washed away again. The bound antibody-peroxidase conjugate can convert a colorless substrate (TMB) to a colored product, which was measured photometrically at 405 nm. Absorption intensity indicates the amount of KBD-B or -D bound.

Skin binding studies were performed on human keratinocytes in microtiter plates as described below.
-Wash twice with PBS / 0.05% Tween20.
Add 1 ml of 1% BSA in PBS and incubate for 1 hour at room temperature with gentle rotation (900 rpm).
Remove the supernatant.
Add 100 μg KBD in PBS (0.05% Tween 20) and incubate for 2 hours at room temperature with gentle swirl (900 rpm).
Remove the supernatant.
-Wash 3 times with PBS / 0.05% Tween20.
Incubate with 1 ml of monoclonal mouse anti-tag His6 antibody for 2-4 hours at room temperature with gentle rotation (900 rpm).
-Wash 3 times with PBS / 0.05% Tween20.
Add peroxidase substrate (1 ml / eppendorf container, see below for composition)
-React until it turns blue (about 90 seconds).
-The reaction was stopped with 100 μl of 2M H ₂ SO ₄ .
-Absorption at 405 nm was measured.

Peroxidase substrate (prepared immediately before):
0.1 ml of TMB solution (42 mM TMB in DMSO)
+10 ml substrate buffer (0.1 M sodium acetate, pH 4.9)
+14.7 μl H ₂ O ₂ (3% concentration).

To characterize the hair binding of KBD-D compared to KBD-B, the following binding assay was performed:
5 mg of hair (human) was cut into 5 mm long pieces and transferred to Eppendorf containers (low protein binding).
・ Add 1 ml of ethanol for degreasing.
· Centrifuged to remove ethanol, wash the hair with H ₂ O.
• Centrifuge and remove supernatant.
Add the keratin-binding domain to be tested (coupled to tags (eg, His ₆ , HA, etc.)) in 1 ml of PBS / 0.05% Tween20 and incubate with gentle swirling for 2 hours at room temperature.
• Centrifuge and remove supernatant.
-Wash 3 times with PBS / 0.05% Tween20.
Monoclonal mouse anti-tag (His6 or HA) antibody (with peroxidase conjugate) (1: 2000 in PBS / 0.05% Tween20) [monoclonal anti-polyhistidine peroxidase conjugate, produced in mice, lyophilized powder, manufactured by Sigma Incubate with 1 ml for 2-4 hours at room temperature with gentle swirling.
-Wash 3 times with PBS / 0.05% Tween20.
Add peroxidase substrate (1 ml / eppendorf container).
・ Proceed with the reaction until it turns blue (90 seconds).
Stop the reaction with 100 μl of 2M H ₂ SO ₄ .
Absorption at 405 nm was measured.

Peroxidase substrate (prepared immediately before):
0.1 ml of TMB solution (42 mM TMB in DMSO)
+10 ml substrate buffer (0.1 M sodium acetate, pH 4,9)
+14.7 μl H ₂ O ₂ (3% concentration).
BSA = bovine serum albumin
PBS = phosphate buffered saline
Tween 20 = polyoxyethylene sorbitan monolaurate, n is about 20
TMB = 3,5,3 ′, 5′-tetramethylbenzidine.

  These results indicate that the protein KBD-D can bind to the hair and can bind more strongly to the skin (see Table 10). Compared to KBD-B (SEQ ID NO: 166), binding of KBD-D (SEQ ID NO: 168) is less affected by washing with SDS solution up to 10% concentration (see Table 10a).

Example 16: Preparation of maleimidocaproic acid
According to the method described in Rich, DH et al. (Rich, DH et al. J. Med. Chem- 1975, 18 (10), 1004-1010), maleimidocaproic acid and its analogs (N = 1 to 4, 7, 10 and 11) were synthesized.

Example 17: Coupling of maleimidocaproic acid with panthenol mediated by carbodiimide-Method A
1.11 g maleimidohexanoic acid and 0.03 g DMAP were added to 3.14 g D-panthenol in 30 ml methylene chloride. At room temperature, 1.05 g of EDC in 25 ml of methylene chloride was added dropwise over 1 hour and the mixture was then stirred at room temperature for 2.5 hours. The resulting solution was washed twice with 25 ml 2N HCl. The organic phase was dried over sodium sulfate and concentrated by evaporation on a rotary evaporator at 40 ° C./4 mbar to give 1.5 g of a slightly yellow viscous residue.
The reaction product was analyzed by HPLC according to the following method.

Column: Waters Symmetry C18, 5μm, 250 * 4.6mm
Eluent A: 0.1% by volume of H ₃ PO ₄ in water, Eluent B: 0.1% by volume of H ₃ PO _{4 in} CH ₃ CN
Gradient (based on eluent B): 0 min (10%), 10 min (100%), 20 min (100%), 22 min (10%)
Flow rate: 1 ml / min, temperature 20 ° C, injection volume 5 μl
Detection: ultraviolet absorption detector (205 nm), BW = 4 nm.

  In this way, the three monoacylated panthenols elute at 7.0, 7.5 and 7.6 minutes, the diacylated panthenols at 9.0, 9.2 and 9.6 minutes and the triacylated panthenol at 10.7 minutes.

  The resulting products include (based on HPLC peak area%) up to 22.2%, 23.1% and 24.7% mono isomers, up to 4.4%, 8.5% and 5.7% diacylated isomers, up to 0.9% Of the triacylated compound (not including the remaining components).

Example 18: Coupling of maleimide-N-hexanoic acid with panthenol after activation as acid chloride-Method B
13.4 g of thionyl chloride is added to 8.0 g of maleimidohexanoic acid in 100 ml of toluene and the mixture is heated at 80 ° C. for 3 hours. The solution was concentrated by evaporation on a rotary evaporator at 70 ° C./3 mbar to give 8.7 g of a yellow liquid. When this was left overnight, it solidified and became pale yellow crystals ( ¹³ C NMR (500 MHz, CDCl ₃ ): 173.4, 170.7 (2C), 134.1 (2C), 46.8, 37.3, 28.0, 25.5, 24.5 ppm ).

  A solution of 8.5 g maleimidocaproyl chloride in 100 ml methylene chloride was added dropwise to 36.9 g D-panthenol and 4.32 g triethylamine in 250 ml methylene chloride over 1 hour at room temperature and the mixture was stirred at room temperature for 2 hours. The solution was washed three times with 60 ml of 2N HCl and twice with 60 ml of water, the organic phase was dried over sodium sulfate and concentrated by evaporation on a rotary evaporator at 40 ° C./2 mbar. 14 g of orange-brown oil was obtained.

  The following product distribution was determined according to the HPLC method described in Example 17 (based on area% of the HPLC peak). 1.3%, 39.5% and up to 32.8% mono isomers, 0.5%, 0.7% and 9.9% diacylated isomers, 0.1% triacylated compounds (excluding remaining components).

Example 19: Coupling maleimidocaproic acid with tocopherol As in Example 17, 2.2 g α-tocopherol and 1 g triethylamine were reacted with 1.5 g MIC-Cl to give α-tocopherol maleimide caproylate (α-tocopherol). maleimidocaproylate) 2g.

Example 20: Coupling maleimidocaproic acid with ascorbic acid As in Example 18, 0.9 g α-tocopherol and 1 g NEt ₃ were reacted with 1.3 g MIC-Cl to give 1.6 g maleimide caproylate ascorbate. Was obtained as a mixture of isomers.

Example 21: Coupling of maleimidocaproic acid with astaxanthin As in Example 18, astaxanthin 0.2g with EDC 0.3g and DMAP 0.01g was reacted with maleimidocaproic acid 0.35g to give maleimidocaproyl-astaxanthin 0.2g Obtained as a mixture of isomers.

The effector linker molecules listed in Table 11 below could be prepared according to Examples 17-21 and could be prepared. All of the effector molecules listed in Table 11 can be preferably coupled in the same manner as the linker molecules described in general formula 1, 1b, 1c, 2, 4 or 5.

Example 22: Effector coupling of panthenol to KBD-B (SEQ ID NO: 166) To couple panthenol via maleimidocaproic acid linker (MIC linker), cysteine of KBD-B (SEQ ID NO: 166) It was used. Therefore, KBD-B (SEQ ID NO: 166) had 4 cysteines. Of these, two cysteines are inside the structure and are not available for effector coupling (recognizable from the crystal structure). The remaining two cysteines near the N-terminus (amino acid positions 14 and 83, see sequence KBD-B (SEQ ID NO: 166)) are available for effector coupling.

  Panthenol-MIC, which can be coupled, was coupled to KBD-B (SEQ ID NO: 166) via at least one of the two free SH groups of cysteine. This resulted in a nucleophilic attack of cysteine on the maleic diimide double bond (FIG. 5).

  After various test batches (see Example 24), an efficient coupling method was established and used for 5 g of the coupling mixture. For this, 1 ml of 10% MIC-panthenol in ethanol is added to 250 ml of phosphate buffer solution of 20 mg / ml KBD-B (pH 7.5) (use a lower concentration of about 1 mg / ml). (KBD-B: MIC-panthenol ratio = 1: 2), the mixture was carefully shaken for 1 hour at room temperature.

Example 23: Effector Coupling of Panthenol to KBD-D (SEQ ID NO: 168) To couple panthenol via maleimidocaproic acid linker (MIC linker), cysteine is KBD-B as well as KBD-B. It can also be used in D (SEQ ID NO: 168). Therefore, KBD-D (SEQ ID NO: 168) has 24 cysteines. Furthermore, cysteine groups that can be coupled can be introduced in a site-specific manner by site-directed mutagenesis.

  Thus, panthenol-MIC, which could be coupled, could be coupled to KBD-D (SEQ ID NO: 168) via at least one free SH group of cysteine. The KDB-D-panthenol effector molecule thus obtained could be used in cosmetic formulations as described in Examples 58-75.

The keratin-binding effector molecules listed in Table 12 and Table 12a below are prepared and can be prepared according to Examples 17-23. All of the effector linker molecules listed there are SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 40, 42, 44, 46, 48, 146, 150, 153, 156, 157, 158, It can be coupled in an analogous manner, preferably to a keratin binding protein according to 160, 162 or 164, particularly preferably to the KBD-D protein represented by SEQ ID NO: 168.

Example 24: Coupling success test (Elman test)
The effector coupling success was monitored by two different tests.

(iii) Elman test that can determine the number of free Cys-SH groups in the protein before and after effector coupling. Here, a marked decrease in free SH groups after coupling indicates a good reaction progression.

(iv) An activity test that can measure the binding of KBD-B to hair with or without coupled panthenol. A good reaction procedure should not reduce the activity of KBD-panthenol compared to uncoupled KBD (see Example 22).

To ensure efficient coupling, various test batches were performed to test different temperatures and KBD-B / panthenol-MIC mixing ratios.
These batches were then tested using the Elman test as follows.
Required materials:
Elman reagent: 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) 4mg / 1ml in 0.1M sodium phosphate buffer
・ 0.1M sodium phosphate buffer (pH8.0)
・ Cysteine solution (cysteine hydrochloride monohydrate 26.3mg / sodium phosphate buffer 100ml)
Solutions must be prepared and prepared immediately before use.

1. In all cases, 25 μl, 50 μl, 100 μl, 150 μl, 200 μl and 250 μl cysteine solutions were pipetted into test tubes (13 × 100 mm) for calibration curves. The protein sample to be measured was poured into separate tubes (volume <= 250 μl). Of the KBD to be tested, an amount of at least 1 mg per reaction mixture was dispensed. In the case of tubes, the total volume was then adjusted to 250 μl with sodium phosphate buffer in each case. If the volume of 250 μl sample was exceeded (for 1 mg of KBD required), this was taken into account when adding 2.5 ml of sodium phosphate buffer in 2.
2. In either case, add 50 μl Ellman's reagent and 2.5 ml sodium phosphate buffer. Mix briefly and incubate for 15 minutes at RT.
3. Measure the absorption at 412nm.
4. Create a calibration curve, plot it, and read the value of the protein sample to be measured.

The Ellman test evaluation (Table 13) shows that 2/3 of the free thiol groups can be coupled to MIC-panthenol when the KBD-B: MIC-panthenol mixing ratio is 1: 2. The temperature does not seem to affect the reaction process very much.

Example 24a: KBD-B-Panthenol Hair Binding Activity
To confirm whether KBD-B also binds to hair even with coupled panthenol, a quantitative binding assay was performed (see FIG. 6). In this test, the hair was first incubated with KBD-B-panthenol to wash away unbound KBD-B-panthenol. Peroxidase was then coupled via the His tag of KBD-B. Unbound peroxidase was washed off again. Bound peroxidase can convert a colorless substrate (TMB) into a colored product, which was measured at 405 nm by photometry. Absorption intensity indicates the amount of KBD-B-pantenol bound. As a comparative sample, KBD-B without panthenol was selected. The results of the test are shown in FIG.

  FIG. 7 shows that the reaction temperature also has no decisive effect on the activity of KBD-B coupled with panthenol. At KBD-B: panthenol mixing ratios up to 1: 2, the activity of the coupled protein remains virtually constant. Only when the mixing ratio exceeds 1: 4, the hair binding activity decreases. Coupling to lysine or cysteine in protein structures that are no longer selective may be involved in this, which may lead to protein unfolding and thus poorer hair coupling .

  Overall, activity and Elman data show that KBD-B-Panthenol coupling proceeds very easily at room temperature with a 1: 2 KBD-B: MIC-Panthenol reaction ratio. -Shows that large quantities of B-panthenol can be produced.

A skin cosmetic preparation according to the present invention comprising a keratin-binding effector molecule (keratin-binding domain represented by SEQ ID NO: 166) produced according to Example 22 coupled to panthenol via a maleimidocaproic acid linker The skin cosmetic preparation according to the invention is described below. In the examples below, the keratin binding effector molecule is referred to as the keratin binding domain MIC-pantenol. The keratin binding domain MIC-panthenol is identified in the examples below as representative of all of the other keratin binding effector molecules described above. One skilled in the art will appreciate that all other specific keratin-binding effector molecules according to Example 22 can also be produced and used in the preparations shown below.

Example 25: Use of KBD in Day Care Emulsions-O / W Type

Preparation: Phases A and B are heated separately to about 80 ° C. Phase B is stirred into Phase A and homogenized. Introduce phase C into the mixed phases A and B with stirring and homogenize again. Cool to about 40 ° C. with stirring, add Phase D, adjust the pH to about 6.5 using Phase E, homogenize and cool to room temperature with stirring.
Note: The formulation is prepared without protective gas. Bottling must be performed on oxygen-impermeable containers, such as aluminum tubes.

Example 26: Use of KBD in protective day cream-O / W type

  Preparation: Phases A and B are heated separately to about 80 ° C. Phase B is stirred into Phase A and homogenized. Phase C is incorporated into mixed phases A and B and homogenized. Cool to about 40 ° C. with stirring. Add Phase D, adjust the pH to about 6.5 using Phase E and homogenize. Cool to room temperature with stirring.

Example 27: Use of KBD in facial cleansing lotion-O / W type

  Preparation: Dissolve phase A. Phase B is stirred into phase A. Incorporate phase C into mixed phases A and B. Dissolve phase D, stir into the mixed phases A, B and C and homogenize. Then stir for 15 minutes.

Example 27a: Use of KBD in Daily Care Body Spray

  Preparation: Phase A ingredients are weighed in and dissolved until clear.

Example 28: Use of KBD in skin care gel

  Preparation: Dissolve Phase A until clear. Swell Phase B and neutralize with Phase C. Stir and introduce phase A into homogenized phase B and homogenize.

Example 29: Use of KBD in After Shave Lotion

  Preparation: Mix Phase A ingredients. Dissolve Phase B, incorporate into Phase A and homogenize.

Example 30: Use of KBD in After Sun Lotion

  Preparation: Mix Phase A ingredients. Phase B is stirred into Phase A with homogenization. Neutralize with phase C and homogenize again.

Example 31: Use of KBD in a sunscreen lotion

  Preparation: Phase A and B ingredients are heated separately to about 80 ° C. Phase B is stirred into Phase A and homogenized. Phase C is heated to about 80 ° C. and stirred into the mixed phases A and B with homogenization. Cool to about 40 ° C. with stirring, add phase D and homogenize again.

Example 32: Use of KBD in sunscreen lotion-O / W type

  Preparation: Heat phase A to about 80 ° C., stir into phase B and homogenize for 3 minutes. Similarly, phase C is heated to 80 ° C. and stirred into the mixed phases A and B with homogenization. Cool to about 40 ° C., stir in Phase D and homogenize again.

Example 33: Use of KBD in sunscreen lotion-O / W type

  Preparation: Heat phase A to about 80 ° C., stir into phase B and homogenize for 3 minutes. Similarly, phase C is heated to 80 ° C. and stirred into the mixed phases A and B with homogenization. Cool to about 40 ° C., stir in Phase D and homogenize again.

Example 34: Use of KBD in foot balsam

  Preparation: Phase A and B ingredients are heated separately to about 80 ° C. Phase B is stirred into Phase A with homogenization. Cool to about 40 ° C. with stirring, add phases C and D, then homogenize briefly. Cool to room temperature with stirring.

Example 35: Use of KBD in W / O emulsion containing bisabolol

  Preparation: Phases A and B are heated separately to about 85 ° C. Phase B is stirred into Phase A and homogenized. Cool to about 40 ° C. with stirring, add phase C and homogenize again briefly. Cool to room temperature with stirring.

List of Formulations for Keratin Binding Domains of the Invention-Hair Care
Example 36: Foam conditioner containing setting agent

  Preparation: Phase A ingredients are weighed together and stirred until everything is dissolved and bottling.

Example 37: Foam conditioner

  Preparation: Phase A ingredients are weighed together, stirred and bottling until everything is dissolved and a clear solution is obtained.

Example 38: Foam conditioner

  Preparation: Phase A ingredients are weighed together, stirred and bottling until everything is dissolved and a clear solution is obtained.

Example 39: Styling foam

  Preparation: Mix Phase A ingredients. Add Phase B ingredients one at a time and dissolve. Bottling with Phase C.

Example 40: Styling foam

  Preparation: Mix Phase A ingredients. Add Phase B ingredients one at a time and dissolve. Bottling with Phase C.

Example 41: Styling foam

  Preparation: Mix Phase A ingredients. Dissolve Phase B ingredients until clear, then Phase B is stirred into Phase A. Adjust pH to 6-7 and bottling with Phase C.

Example 42: Styling foam

  Preparation: Mix Phase A ingredients. Add Phase B ingredients one at a time and dissolve. Phase C is dissolved in a mixture of A and B, then the pH is adjusted to 6-7. Bottling with Phase D.

Example 43: Styling foam

  Preparation: Mix Phase A ingredients. Add Phase B ingredients one at a time and dissolve. Phase C is dissolved in a mixture of A and B, then the pH is adjusted to 6-7. Bottling with Phase D.

Example 44: Styling foam

Styling foam

  Preparation: Dissolve phase A. Phase B is metered into Phase A and dissolved until clear. Adjust pH to 6-7 and bottling with Phase C.

Example 45: Styling foam

  Preparation: Dissolve phase A. Phase B is metered into Phase A and dissolved until clear. Adjust pH to 6-7 and bottling with Phase C.

Example 46: Styling foam

  Preparation: Dissolve phase A. Phase B is metered into Phase A and dissolved until clear. Adjust pH to 6-7 and bottling with Phase C.

Example 47: Styling foam

  Preparation: Mix Phase A ingredients. Add Phase B ingredients one at a time and dissolve. Bottling with Phase C.

Example 48: Care shampoo

  Preparation: Mix and dissolve Phase A ingredients. Adjust the pH to 6-7 with citric acid.

Example 49: Shower gel

  Preparation: Mix and dissolve Phase A ingredients. Adjust the pH to 6-7 with citric acid.

Example 50: Shampoo

  Preparation: Mix and dissolve Phase A ingredients. Adjust the pH to 6-7 with citric acid.

Example 51: Shampoo

  Preparation: Phase A ingredients are metered in and dissolved. Adjust the pH to 6-7. Add Phase B and heat to about 50 ° C. Cool to room temperature with stirring.

Example 52: Moisturizing body care cream

  Preparation: Phases A and B are heated separately to about 80 ° C. Pre-homogenize phase B for a short time, then stir introduce phase B into phase A and homogenize again. Cool to about 40 ° C., add phase C and homogenize completely again. Adjust the pH to 6-7 with citric acid.

Example 53: Moisturizing body care cream

  Preparation: Phases A and B are heated separately to about 80 ° C. Phase B is stirred into Phase A and homogenized. Cool to about 40 ° C. with stirring, add phase C and homogenize again. Allow to cool to room temperature with stirring.

Example 54: Liquid makeup-O / W type

  Preparation: Phases A and B are heated separately to about 80 ° C. Phase B is stirred into Phase A and homogenized. Cool to about 40 ° C. with stirring, add phases C and D, and homogenize completely again. Allow to cool to room temperature with stirring.

Example 55
A skin cosmetic preparation according to the present invention comprising a keratin-binding effector molecule produced according to Example 23 (keratin-binding domain represented by SEQ ID NO: 166) coupled to panthenol via a maleimidocaproic acid linker. This is explained below. The specific keratin binding effector molecule is referred to in the examples below as keratin binding domain MIC-pantenol.

Specific keratin-binding effector molecules are used as an aqueous solution with a concentration of about 5% by weight. The following data are parts by weight.

Example 56
100 mg of the day care emulsion of Example 19 containing 5% strength by weight keratin binding domain MIC-panthenol (with water as 100) (and no keratin binding domain MIC-panthenol as placebo) Also in the case of applying to the inside of the forearm. After 30 minutes, 4 out of 5 subjects found a noticeable better feel on the skin inside the forearm treated with the keratin binding domain MIC-panthenol. All five subjects felt that the side treated with the active ingredient according to the present invention was much moist, ie less dry.

Example 57
A cosmetic sunscreen preparation comprising at least one inorganic pigment, preferably zinc oxide and / or titanium dioxide, and a combination of organic UV-A and UV-B filters is described in the following formulation.

  The formulations specified below are prepared by conventional methods known to those skilled in the art.

The content of the keratin-binding effector molecule prepared according to Example 23 (keratin-binding domain represented by SEQ ID NO: 166) coupled to panthenol via a maleimidocaproic acid linker represents 100% of the active ingredient. The active ingredient according to the invention can be used in pure form, otherwise in the form of an aqueous solution. In the case of aqueous solutions, the content of demineralized water in a particular formulation must be adjusted.

  A skin cosmetic preparation according to the present invention comprising a keratin-binding effector molecule produced according to Example 23 (keratin-binding domain represented by SEQ ID NO: 168) coupled to panthenol via a maleimidocaproic acid linker. This is explained below. The keratin-binding effector molecule is referred to as keratin-binding domain MIC-pantenol in the examples below. The keratin binding domain MIC-panthenol is identified in the examples below as representative of all of the other keratin binding effector molecules described above. One skilled in the art will appreciate that all other specific keratin-binding effector molecules according to Example 23 can also be generated and used in the preparations shown below.

Example 58: Use of KBD in Day Care Emulsions-O / W Type

Preparation: Phases A and B are heated separately to about 80 ° C. Phase B is stirred into Phase A and homogenized. Introduce phase C into the mixed phases A and B with stirring and homogenize again. Cool to about 40 ° C. with stirring, add Phase D, adjust the pH to about 6.5 using Phase E, homogenize and cool to room temperature with stirring.
Note: The formulation is prepared without protective gas. Bottling must be performed on oxygen-impermeable containers, such as aluminum tubes.

Example 59: Use of KBD in Protective Day Cream-O / W Type

  Preparation: Phases A and B are heated separately to about 80 ° C. Phase B is stirred into Phase A and homogenized. Phase C is incorporated into mixed phases A and B and homogenized. Cool to about 40 ° C. with stirring. Add Phase D, adjust the pH to about 6.5 using Phase E and homogenize. Cool to room temperature with stirring.

Example 60: Use of KBD in facial cleansing lotion-O / W type

  Preparation: Dissolve phase A. Phase B is stirred into phase A. Incorporate phase C into mixed phases A and B. Dissolve phase D, stir into the mixed phases A, B and C and homogenize. Then stir for 15 minutes.

Example 60a: Use of KBD in Daily Care Body Spray

  Preparation: Phase A ingredients are weighed in and dissolved until clear.

Example 61: Use of KBD in skin care gel

  Preparation: Dissolve Phase A until clear. Swell Phase B and neutralize with Phase C. Stir and introduce phase A into homogenized phase B and homogenize.

Example 62: Use of KBD in After Shave Lotion

  Preparation: Mix Phase A ingredients. Dissolve Phase B, incorporate into Phase A and homogenize.

Example 63: Use of KBD in After Sun Lotion

  Preparation: Mix Phase A ingredients. Phase B is stirred into Phase A with homogenization. Neutralize with phase C and homogenize again.

Example 64: Use of KBD in a sunscreen lotion

  Preparation: Phase A and B ingredients are heated separately to about 80 ° C. Phase B is stirred into Phase A and homogenized. Phase C is heated to about 80 ° C. and stirred into the mixed phases A and B with homogenization. Cool to about 40 ° C. with stirring, add phase D and homogenize again.

Example 65: Use of KBD in sunscreen lotion-O / W type

  Preparation: Heat phase A to about 80 ° C., stir into phase B and homogenize for 3 minutes. Similarly, phase C is heated to 80 ° C. and stirred into the mixed phases A and B with homogenization. Cool to about 40 ° C., stir in Phase D and homogenize again.

Example 66: Use of KBD in Sunscreen Lotion-O / W Type

  Preparation: Heat phase A to about 80 ° C., stir into phase B and homogenize for 3 minutes. Similarly, phase C is heated to 80 ° C. and stirred into the mixed phases A and B with homogenization. Cool to about 40 ° C., stir in Phase D and homogenize again.

Example 67: Use of KBD in foot balsam

  Preparation: Phase A and B ingredients are heated separately to about 80 ° C. Phase B is stirred into Phase A with homogenization. Cool to about 40 ° C. with stirring, add phases C and D, then homogenize briefly. Cool to room temperature with stirring.

Example 68: Use of KBD in a W / O emulsion containing bisabolol

  Preparation: Phases A and B are heated separately to about 85 ° C. Phase B is stirred into Phase A and homogenized. Cool to about 40 ° C. with stirring, add phase C and homogenize again briefly. Cool to room temperature with stirring.

List of Formulations for Keratin Binding Domains of the Invention-Hair Care
Example 69: Shower gel

  Preparation: Mix and dissolve Phase A ingredients. Adjust the pH to 6-7 with citric acid.

Example 70: Moisturizing body care cream

  Preparation: Phases A and B are heated separately to about 80 ° C. Pre-homogenize phase B for a short time, then stir introduce phase B into phase A and homogenize again. Cool to about 40 ° C., add phase C and homogenize completely again. Adjust the pH to 6-7 with citric acid.

Example 71: Moisturizing body care cream

  Preparation: Phases A and B are heated separately to about 80 ° C. Phase B is stirred into Phase A and homogenized. Cool to about 40 ° C. with stirring, add phase C and homogenize again. Allow to cool to room temperature with stirring.

Example 72: Liquid makeup-O / W type

  Preparation: Phases A and B are heated separately to about 80 ° C. Phase B is stirred into Phase A and homogenized. Cool to about 40 ° C. with stirring, add phases C and D, and homogenize completely again. Allow to cool to room temperature with stirring.

Example 73
A skin cosmetic preparation according to the present invention comprising a keratin-binding effector molecule produced according to Example 23 (keratin-binding domain represented by SEQ ID NO: 168) coupled to panthenol via a maleimidocaproic acid linker. This is explained below. The specific keratin binding effector molecule is referred to in the examples below as keratin binding domain MIC-pantenol.

The specific keratin-binding effector molecule is used as an aqueous solution with a concentration of about 5% by weight. The following data are parts by weight.

Example 74
100 mg of the day care emulsion of Example 19 containing 5% strength by weight keratin binding domain MIC-panthenol (with water as 100) (and no keratin binding domain MIC-panthenol as placebo) Also in the case of applying to the inside of the forearm. After 30 minutes, 4 out of 5 subjects found a noticeable better feel on the skin inside the forearm treated with the keratin binding domain MIC-panthenol. All five subjects felt that the side treated with the active ingredient according to the present invention was much moist, ie less dry.

Example 75
A cosmetic sunscreen preparation comprising at least one inorganic pigment, preferably zinc oxide and / or titanium dioxide, and a combination of organic UV-A and UV-B filters is described in the following formulation.

  The formulations specified below are prepared by conventional methods known to those skilled in the art.

The content of keratin-binding effector molecule (keratin-binding domain represented by SEQ ID NO: 168) prepared according to Example 23, coupled to panthenol via a maleimidocaproic acid linker represents 100% of the active ingredient. The active ingredient according to the invention can be used in pure form, otherwise in the form of an aqueous solution. In the case of aqueous solutions, the content of demineralized water in a particular formulation must be adjusted.

It is a vector map of pQE30-KBD-B for expressing keratin binding domain B. It is a vector map of pLib15 for expressing keratin binding domain B. It is a vector map of pLib16 for expressing keratin binding domain B. It is a vector map of pLib19 for expressing keratin binding domain B. FIG. 6 shows the coupling reaction of activated panthenol to KBD-B to cysteine (effector linker molecule 137 according to Table 11). It is a schematic diagram which shows a quantitative KBD activity test. The His tag antibody-peroxidase conjugate couples to the KBD that is bound to the skin or hair via the His tag. Peroxidase catalyzes the color reaction and its final product is yellow, which can be measured photometrically. 2 is a graph showing photometric values of quantitative hair activity tests for various KBD-B: panthenol mixing ratios at reaction temperatures of 22 ° C. and 4 ° C. 1 shows a KBD-D expression vector for protein production in E. coli.

Claims (19)

The effector molecule (i) carrying at least one hydroxy or amino function is selected on the keratin-binding polypeptide (ii) from the group consisting of thioester bond, ester bond, thioether bond, ether bond and amide bond A keratin-binding effector molecule by coupling using a linker molecule (iii) having at least two coupling functional groups capable of binding
(a) in the first coupling step, the effector molecule (i) is first bound to the linker molecule (iii) via an ester bond or an amide bond;
(b) in a further coupling step, the reaction product from (a) is coupled to the keratin-binding polypeptide (ii) via the still free coupling functional group of the linker molecule (iii) Method.   The method according to claim 1, wherein the coupling between the linker molecule (iii) and the effector molecule (i) described in (a) is a carbodiimide, anhydride or acid chloride mediated esterification reaction. .   The method according to claim 1 or 2, wherein the effector molecule (i) is selected from the group consisting of pigments, photoprotective agents, vitamins, provitamins, carotenoids, antioxidants and peroxide degrading agents.   The method according to any one of claims 1 to 3, wherein the keratin-binding polypeptide (ii) has binding affinity for keratin of human skin, hair or nails. The keratin-binding polypeptide (ii) used is
(a) SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 146, 150, 153 , 156, 157, 158, 160, 162, 164, 166, 168 or 170, or
(b) SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 146, 150, 153 156, 157, 158, 160, 162, 164, 166, 186 or 170, which corresponds to a polypeptide which is at least 40% identical and capable of binding to keratin with at least one of the sequences represented by 5. The method according to any one of 4 above. The keratin-binding polypeptide (ii) used is
(a) SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 146, 150, 153 156, 157, 158, 160, 162, 164, 166, 168 or 170, a nucleic acid molecule encoding a polypeptide comprising the sequence shown in
(b) SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 145, 149, 152 159, 161, 163, 165, 167 or 169, a nucleic acid molecule comprising at least one polynucleotide of the sequence shown in
(c) SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 146, 150, 153 , 156, 157, 158, 160, 162, 164, 166, 168 or 170, a nucleic acid molecule encoding a polypeptide represented by the sequence,
(d) SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 145, 149, 152 , 159, 161, 163, 165, 167 or 169, a nucleic acid molecule having a nucleic acid sequence corresponding to at least one of the sequences represented by, or SEQ ID NO: 2, derived from this nucleic acid molecule by substitution, deletion or insertion 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, A poly that is at least 40% identical to at least one of the sequences represented by 138, 140, 146, 150, 153, 156, 157, 158, 160, 162, 164, 166, 168 or 170 and is capable of binding to keratin. A nucleic acid molecule encoding a peptide,
(e) a nucleic acid molecule encoding a polypeptide recognized by a monoclonal antibody against the polypeptide encoded by the nucleic acid molecule according to (a)-(c),
(f) a nucleic acid molecule encoding a keratin-binding protein that hybridizes with the nucleic acid molecule according to (a) to (c) under stringent conditions,
(g) Keratin, which can be isolated from a DNA bank under stringent hybridization conditions using at least 15 nucleotides of the nucleic acid molecule according to (a) to (c) or a partial fragment thereof as a probe A nucleic acid molecule encoding a binding protein,
(h) SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 146, 150, 153 , 156, 157, 158, 160, 162, 164, 166, 168 or 170, at least one nucleic acid selected from the group consisting of nucleic acid molecules that can be generated by back-translating one of the amino acid sequences shown in 6. A method according to any one of claims 1 to 5, encoded by a nucleic acid molecule comprising a molecule.   The method according to any one of claims 1 to 6, wherein the linker molecule (iii) has at least two different coupling functional groups.   The method according to claim 7, wherein the linker molecule (iii) has a maleimide group. The linker molecule (iii) used is of the formula 1:

9. The method according to claim 8, which is a carboxylic acid-supported maleimide (wherein “n” corresponds to an integer of 0 to 40).   The method according to claim 9, wherein the linker molecule (iii) is maleimidocaproic acid. (h) the keratin-binding polypeptide used is SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 , 38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70,72,74,76,78,80,82,84,86 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136 138, 140, 146, 150, 153, 156, 157, 158, 160, 162, 164, 166, 168 or 170, including one of the sequences
(i) the linker molecule (iii) used is maleimidocaproic acid, and
(j) The effector molecule (i) is selected from the group consisting of pantothenic acid, panthenol, an ester of panthenol, an ether of panthenol, and a cation-derivatized panthenol. The method according to item.   The effector molecule (i) is indirectly coupled to the keratin-binding polypeptide (ii) via a linker molecule (iii) provided that maleimide is a linker during the production of the keratin-binding effector molecule Keratin that is not used as molecule (iii), the polypeptide represented by SEQ ID NO: 166 is not used as keratin-binding polypeptide (ii), and no fluorescent dye is used as effector molecule (ii) Binding effector molecule.   A keratin-binding effector molecule produced according to claim 11.   Use of the keratin-binding effector molecule according to claims 12 and 13 or the keratin-binding effector molecule produced according to claims 1-11 in a skin cosmetic.   15. The skin cosmetic composition according to claim 14, wherein the skin cosmetic composition is a skin protection composition, skin care composition, skin cleansing composition, hair protection composition, hair care composition, hair cleansing composition, hair colorant or decorative cosmetic. use. A method of applying a skin cosmetic active effector molecule to the skin, hair, and / or fingernails and toenails,
(k) coupling the skin cosmetic active ingredient to a keratin-binding polypeptide; and
(l) applying the keratin-binding effector molecule according to (k) to the skin, hair and / or fingernails and toenails as a component of a skin cosmetic preparation;
The above method. A method for increasing the residence time of a skin cosmetic active ingredient on the skin, hair, and / or fingernails or toenails,
(m) coupling the skin cosmetic active ingredient to a keratin-binding polypeptide;
(n) applying the keratin-binding effector molecule according to (m) to the skin, hair, and / or fingernails or toenails as a component of a skin cosmetic preparation; and
(o) the active ingredient binds indirectly to the skin, hair, and / or fingernails or toenails via a keratin binding domain;
The above method. Formula 2:

Wherein “n” is an integer from 0 to 40.   A skin cosmetic comprising the keratin-binding effector molecule according to claim 12 or 13, or the keratin-binding effector molecule produced according to claims 1-11.

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